T. Tselios

In vivo evaluation and in vitro metabolism of leuprolide in mice--mass spectrometry-based biomarker measurement for efficacy and toxicity.

The study of pharmacologically active peptides is central for the understanding of cancer and the development of novel therapeutic approaches. In this context, both qualitative and quantitative determination of bioactive peptides in biological fluids/tissues and their effect on endogenous factors (e.g. hormones) are of great importance. A mass spectrometry-based approach was developed and applied towards the measurement of leuprolide, a peptide drug for the treatment of prostate cancer, in mouse plasma. High-pressure liquid chromatography coupled to a hybrid quadrupole linear ion trap (QqLIT) mass spectrometer, a platform that combines the benefits of triple QqLIT instruments, was employed for the study. Using the described methodology, we established that picomolar concentrations of leuprolide could be measured in mouse plasma (limit of quantification of 0.1 ng/ml). In order to optimize pharmacokinetic properties of analogs of leuprolide, a facile in vivo mouse model was developed and leuprolide concentrations were determined in mouse plasma following intraperitoneal administration. In the same animal model, we demonstrated the versatility of the described MS-based approach by the determination of plasma concentrations of testosterone, an established biomarker for the treatment of prostate cancer. Following dosing with leuprolide, circulating testosterone was increased significantly in comparison to vehicle-treated mice. Finally, in vitro metabolism of leuprolide was evaluated by incubation of leuprolide with mouse kidney membranes, followed by identification of major metabolites by MS. Such studies provide the framework for future evaluation of novel leuprolide analogs with potential therapeutic advantages.

Structural Requirements for Binding of Myelin Basic Protein (MBP) Peptides to MHC II: Effects on Immune Regulation

Confronting Multiple Sclerosis requires as an underlying step the manipulation of immune response through modification of Myelin Basic Protein peptides. The aim is to design peptidic or nonpeptidic molecules that compete for recognition of self-antigens at the level of antigen presentation. The rational approach is to substitute residues that serve as anchors for the T-Cell Receptor with others that show no binding at all, and those that serve as Major Histocompatibility Complex II anchors with others that present increased binding affinity. The resulting structure, hence, retains normal or increased MHC II binding properties, but fails to activate disease-inducing T-cells. This rational design can only be achieved by identifying the structural requirements for binding of the natural peptide to MHC II, and the anchor residues with their corresponding specific pockets in the binding groove. The peptide-MHC II complex then interacts with the TCR; thus, an additional way to trigger the desired immune response is to alter secondary anchor residues as well as primary ones. In this review, the structural requirements for binding of MBP peptides to MHC II are presented, as are the mechanism and key features for TCR recognition of the peptide-MHC II complex.

Design and synthesis of small semi-mimetic peptides with immunomodulatory activity based on Myelin Basic Protein (MBP)

SummaryExperimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP). Analogs of these disease-associated peptides have been identified with disease progression upon coimmunization. Usage of peptides, with disease-specific immunomodulatory capacity in vivo is limited, however, due to their sensitivity to proteolytic enzymes. Alternative approaches include the development of mimetic molecules which maintain the biological function of an original peptide, yet are stable and able to elicit their response in pharmacological quantities. A novel technique was employed to design a series of semi-mimetic peptides, based on the guinea pig MBP72–85 peptide used to induce EAE in Lewis rats. We used isonipecotic (iNip) and aminocaproic (Acp) acids as templates. Acp-MBP72–85 peptide derived analogues were effective in inducing EAE compared to iNip-peptide analogues which were ineffective at 350μg. These findings suggest that the design and synthesis of semi-mimetic peptide molecules with immunomodulatory potential is possible and that eventually these molecules may form the basis for the development of novel and more effective disease-specific therapeutic agents.

Structural comparison between type I and type II antagonists : Possible implications in the drug design of AT1 antagonists

Analogues of sarilesin (type I AT1 antagonists), and sarmesin (type II AT1 antagonists) with homoserine (hSer) at position 8 were prepared and bioassayed. The presence of a Tyr4-Ile5-His6 bend found in sarmesin but not in sarilesin was identified. The obtained results coupled with conformational analysis studies, using a combination of NMR spectroscopy and computational chemistry, propose important conformational and stereoelectronic properties for agonist and antagonist activity at AT1 receptors.

Conformational analysis of the ΜΒΡ83–99 (Phe91) and ΜΒΡ83–99 (Tyr91) peptide analogues and study of their interactions with the HLA-DR2 and human TCR receptors by using Molecular Dynamics

The two new synthetic analogues of the MBP83–99 epitope substituted at Lys91 (primary TCR contact) with Phe [MBP83–99 (Phe91)] or Tyr [MBP83–99 (Tyr91)], have been structurally elucidated using 1D and 2D high resolution NMR studies. The conformational analysis of the two altered peptide ligands (APLs) has been performed and showed that they adopt a linear and extended conformation which is in agreement with the structural requirements of the peptides that interact with the HLA-DR2 and TCR receptors. In addition, Molecular Dynamics (MD) simulations of the two analogues in complex with HLA-DR2 (DRA, DRB1*1501) and TCR were performed. Similarities and differences of the binding motif of the two analogues were observed which provide a possible explanation of their biological activity. Their differences in the binding mode in comparison with the MBP83–99 epitope may also explain their antagonistic versus agonistic activity. The obtained results clearly indicate that substitutions in crucial amino acids (TCR contacts) in combination with the specific conformational characteristics of the MBP83–99 immunodominant epitope lead to an alteration of their biological activity. These results make the rational drug design intriguing since the biological activity is very sensitive to the substitution and conformation of the mutated MBP epitopes.

A comparative SAR study of thrombin receptor derived non peptide mimetics: importance of phenyl/guanidino proximity for activity

SummaryThrombin, the most potent physiological platelet agonist interacts with cells through a specific G protein-coupled receptor which has been cloned and sequenced. Synthetic thrombin receptor peptides (TRAPS) comprising the first 5 amino acids (SFLLR and SFLLR-NH2) of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombins proteolytic activity were found to cause full platelet aggregation. During the screening of novel thrombin receptor derived non-peptide mimetics in the platelet aggregation assay we found that 1-phenylacetyl-4-(6-guanidohexanoyl)-piperazine (1) and 1-(6-guanidohexanoyl)-4-(phenylacetylamidomethyl)-piperidine (2) exertedin vitro antagonist activities (56% and 40% correspondingly) as it is depicted by the platelet aggregation assay. Using Molecular Modeling, the synthetic compounds were overlayed with SFFLR. All three superimposed low energy structures had Phe and Arg aminoacids in spatial close proximity. The superimposition results revealed that1 resembled more the stereoelectronic environment of SFLLR than2. This difference may be related to their different antagonist efficacy.

Role of myelin basic protein epitope MBP74-85 in experimental autoimmune encephalomyelitis: Elaboration of agonist and antagonist motifs

Conformational properties of the myelin basic protein epitope QKSQRSQDENPV (MBP74‐85) which can initiate experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis, were investigated by semiempirical methods. Energy calculations were carried out on the full MBP74‐85 autoantigen and the antagonist analog [Ala81]MBP74‐85. These studies have revealed a low energy cyclic conformation for MBP74‐85 which is characterized by an agonist motif comprising an interaction of the sidechains of Arg78 and Asp81 of MBP74‐85. Disruption of this agonist motif by removal of the residue 81 carboxylate, as in the antagonist [Ala81]MBP74‐85, invokes a compensatory rearrangement of the molecule resulting in interaction of the Arg78 sidechain with the sidechain of Glu82 together with Lys75. This antagonist motif, comprising guanidino, amino, and carboxylate groups, has been reproduced previously in semimimetic peptides having the general structure Ser‐Arg‐LINKER‐Glu‐NH2 (where LINKER = one or more residues of aminocaproic acid or isonipecotic acid), which have preferred conformations characterized by interaction of the carboxylate with both the guanidino and amino groups in a similar manner to the antagonist motif in [Ala81]MBP74‐85. However, in EAE assays these small semimimetics turned out to be partial agonists, i.e., molecules with structures between agonists and antagonists. These findings provide insight into the design of small molecule (orally active) autoantigen antagonists for the treatment of autoimmune diseases such as MS. Drug Dev. Res. 48:1–5, 1999.

Design and synthesis of non peptide mimetics of the immunodominant 83-99 myelin basic protein epitope (MBP83-99)

Reference EPFL-CONF-184380doi:10.1002/psc.2449View record in Web of Science Record created on 2013-02-27, modified on 2017-05-12No abstract is available for this article.

Molecular modeling of GnRH analogues in DMSO solution using nuclear magnetic resonance (NMR) and molecular dynamics (MD) [Abstract]

Gonadotropin Releasing Hormone (pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH2, GnRH) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. A large number of linear GnRH analogues has been synthesized and tested for several medical uses. Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-(D)Leu-Leu-Arg-Pro-NHEt, LPA) is a potent GnRH agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. Despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of Leuprolide are known. Moreover, non crystallographic data is available for the receptor of GnRH (G protein-coupled receptor). The aim of this study was to characterize the conformation of Leuprolide and its modifi ed linear analogue (pGlu-His-Trp-Ser-Tyr(OMe)-(D)Leu-Leu- Arg-Aze-NHEt) in DMSO solution (which simulates better the receptor environment) using Nuclear Magnetic Resonance (NMR) and Molecular Modeling techniques. By using both NMR and Molecular Modeling we have characterized the secondary structural preferences of these GnRH analogues.

Design and synthesis of two potent amide linked cyclic analogs of the hormone angiotensin II confirm the importance of ring cluster and relay system in its possible bioactive conformation

Panagiota Roumelioti, Ludmila Polevaya, Demetrios V. Vlahakos, Thomas M. Mavromoustakos, Antonios Kolocouris, Theodoros Tselios, and John M. Matsoukas Department of Chemistry, University of Patras, Patras 26500, Greece; 2 Latvian Institute of Organic Synthesis, 21 Aizkraukles, Riga LV-1006, Latvia; Onassis Cardiac Surgery Center, 356 Sygrou Ave, Athens 17674, Greece; and Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Vasileos Constantinou 48, Athens 11635, Greece.