Refinement of the gonadotropin releasing hormone receptor I homology model by applying molecular dynamics

Sexual maturation of human cells in ovaries and prostate is linked to the biochemical cascade initiated by the activation of cell receptors through the binding of Gonadotropin Releasing Hormone (GnRH). The GnRH receptors (GnRHR) are part of the rhodopsin G-protein coupled receptor (GPCR) family and consist of seven trans–membrane helical domains connected via extra– and intra–cellular segments. The GnRH–GnRHR complex has been implicated in various forms of prostate and ovarian cancer. The lack of any structural data about the GnRH receptor impedes the design of antagonists for use in cancer treatment. The aim of the study is to devise a model of GnRHR to be used further for the design of improved peptide/non-peptide GnRH analogues and, to our knowledge provide new structural information regarding the extracellular loop 2 (ECL2) that acts a regulator of ligand entry to GnRHR. The common structural characteristics, of the members of the rhodopsin family of GPCRs, have been employed for the construction of a homology model for GnRHR. Structural information from the human β2–adrenergic receptor, as well as rhodopsins have been used in order to create a theoretical model for GnRHR. Furthermore, molecular dynamics (MD) simulations have been employed for the refinement of the model and to explore the impact of the bilayer membrane in GnRHR conformation

Design, synthesis and evaluation of peptide and non-peptide analogues of gonadotropin releasing hormone GnRH

Gonadotropin Releasing Hormone (GnRH or LHRH) is a decapeptide responsible for the control and secretion of the gonadotropin hormones (LH and FSH) and therefore regulates the reproductive axis. Altered GnRH peptide analogues have been used for the treatment of fertility problems and hormone-dependent cancer. Anticancer drugs are powerful and very effective but with serious side effects. The development of a novel targeted cancer treatment with reduced side effects is of great interest and a challenge for the pharmaceutical industry.In this thesis, design and development of GnRH peptide analogues with a D-amino acid in position 6 as well as with a modified C-terminal based on Leuprolide are reported. The final synthesized peptide analogues contained a thiol group on the side chain of the amino acid in position 6.Moreover, design and synthesis of anthraquinone analogues as well as development of a modified mitoxantrone analogue, a commercially available drug, were carried out. The final compounds included also a thiol group. The conjugation of the synthesized compounds with the altered GnRH peptide analogues was accomplished with the formation of a disulfide bond.The target of this thesis was the binding of the peptide analogue to the GnRH receptor expressed in cancer cells and the subsequent release of the cytotoxic compound (anthraquinone/mitoxantrone analogue) via the reduction of the disulfide bond by the thioredoxin system.Finally, conformational studies of the peptide analogue [DLys6, Pro9-NHEt]GnRH and the respective conjugates with the anthraquinone analogues were carried out, using Molecular Modeling techniques.Η εκλυτική ορμόνη των γοναδοτροπινών (GnRH ή LHRH) είναι ένα δεκαπεπτίδιο υπεύθυνο για τον έλεγχο και την απελευθέρωση των γοναδοτροπινών ορμονών LH και FSH και κατ’ επέκταση για τη ρύθμιση της λειτουργίας του αναπαραγωγικού άξονα. Τροποποιημένα πεπτιδικά ανάλογα της GnRH χρησιμοποιούνται για τη θεραπεία προβλημάτων γονιμότητας αλλά και του ορμονοεξαρτώμενου καρκίνου, καθώς οι υποδοχείς της GnRH εκφράζονται σε μεγάλο ποσοστό σε διαφορετικούς τύπους καρκινικών κυττάρων. Τα αντικαρκινικά φάρμακα που χρησιμοποιούνται είναι ισχυρά και αποτελεσματικά, αλλά με σοβαρές παρενέργειες. Η ανάπτυξη μίας καινοτόμου στοχευμένης θεραπείας με καλύτερη αποτελεσματικότητα και μειωμένες παρενέργειες παρουσιάζει μεγάλο ενδιαφέρον και αποτελεί ερευνητική πρόκληση.Στα πλαίσια της διδακτορικής διατριβής πραγματοποιήθηκε σχεδιασμός και σύνθεση πεπτιδικών αναλόγων της GnRH με χρησιμοποίηση D-αμινοξέων στη θέση 6 καθώς και με τροποποιήσεις στο C-τελικό άκρο, σύμφωνα με το Leuprolide. Τα τελικά πεπτιδικά ανάλογα που συντέθηκαν είχαν τροποποιηθεί κατάλληλα, ώστε να εισαχθεί μία θειολομάδα στην πλευρική αλυσίδα του αμινοξέος στη θέση 6. Επίσης, πραγματοποιήθηκε σχεδιασμός και σύνθεση αναλόγων ανθρακινόνης καθώς και ανάπτυξη τροποποιημένου αναλόγου της εμπορικά διαθέσιμης μιτοξανδρόνης, με σκοπό επίσης την εισαγωγή μίας θειολομάδας. Ακολούθησε η σύζευξη των συντεθειμένων αναλόγων με τα τροποποιημένα πεπτιδικά ανάλογα της GnRH μέσω δισουλφιδικού δεσμού. Απώτερος στόχος της διδακτορικής διατριβής ήταν η δέσμευση του πεπτιδικού αναλόγου στον υποδοχέα της GnRH των καρκινικών κυττάρων και η απελευθέρωση της κυτταροτοξικής ένωσης (ανάλογο ανθρακινόνης/μιτοξανδρόνης) μέσω αναγωγής του δισουλφιδικού δεσμού με την συμμετοχή του συστήματος της θειορεδοξίνης. Τέλος, έγινε διαμορφωτική μελέτη του πεπτιδικού αναλόγου [DLys6, Pro9-NHEt]GnRH καθώς και των αντίστοιχων συζευγμάτων του πεπτιδίου με τα ανάλογα ανθρακινόνης με χρήση τεχνικών Μοριακής Μοντελοποίησης

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87–99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis

In this report, amide-linked cyclic peptide analogues of the 87–99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87–99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72–85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87–99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91–99)[Ala96]MBP87–99, cyclo(87–99)[Ala91,96]MBP87–99 and cyclo(87–99)[Arg91, Ala96]MBP87–99, but not wild-type linear MBP87–99, strongly inhibited MBP72–85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87–99)[Arg91, Ala96]MBP87–99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction

Characterization of Asparagine Deamidation in Immunodominant Myelin Oligodendrocyte Glycoprotein Peptide Potential Immunotherapy for the Treatment of Multiple Sclerosis

Mannan (polysaccharide) conjugated with a myelin oligodendrocyte glycoprotein (MOG) peptide, namely (KG)5MOG35–55, represents a potent and promising new approach for the immunotherapy of Multiple Sclerosis (MS). The MOG35–55 epitope conjugated with the oxidized form of mannan (poly-mannose) via a (KG)5 linker was found to inhibit the symptoms of MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) in mice using prophylactic and therapeutic vaccinated protocols. Deamidation is a common modification in peptide and protein sequences, especially for Gln and Asn residues. In this study, the structural solution motif of deaminated peptides and their functional effects in an animal model for MS were explored. Several peptides based on the MOG35–55 epitope have been synthesized in which the Asn53 was replaced with Ala, Asp, or isoAsp. Our results demonstrate that the synthesized MOG peptides were formed to the deaminated products in basic conditions, and the Asn53 was mainly modified to Asp. Moreover, both peptides (wild type and deaminated derivative) conjugated with mannan (from Saccharomyces cerevisiae) independently inhibited the development of neurological symptoms and inflammatory demyelinating spinal cord lesions in MOG35–55-induced EAE. To conclude, mannan conjugated with a deamidated product did not affect the efficacy of the parent peptide