Theodore Tselios

University of Patras, Rhion, West Greece, Greece

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Publications (64)197 Total impact

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    ABSTRACT: A fast and efficient microwave-assisted solid-phase peptide synthesis protocol utilizing the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of microwave irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the microwave irradiation, the degree of resin substitution during the coupling of the first fourth amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    No preview · Article · Aug 2015 · Biopolymers
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    ABSTRACT: Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and it has been established that autoreactive T helper (Th) cells play a crucial role in its pathogenesis. Myelin basic protein (MBP) epitopes are major autoantigens in MS, and the sequence MBP87-99 is an immunodominant epitope. We have previously reported that MBP87-99 peptides with modifications at principal T-cell receptor (TCR) contact sites suppressed the induction of EAE symptoms in rats and SJL/J mice, diverted the immune response from Th1 to Th2 and generated antibodies that did not cross react with the native MBP protein. In this study, the linear and cyclic analogs of the MBP87-99 epitope, namely linear (Ala91,Ala96)MBP87-99 (P2) and cyclo(87-99)(Ala91,Ala96)MBP87-99 (P3), were evaluated for their binding to HLA-DR4, stability to lysosomal enzymes, their effect on cytokine secretion by peripheral blood mononuclear cells (PBMC) derived from MS patients or healthy subjects (controls), and their effect in rat EAE. P1 peptide (wild-type, MBP87-99) was used as control. P2 and P3 did not alter significantly the cytokine secretion by control PBMC, in contrast to P1 that induced moderate IL-10 production. In MS PBMC, P2 and P3 induced the production of IL-2 and IFN-γ, with a simultaneous decrease of IL-10, whereas P1 caused a reduction of IL-10 secretion only. The cellular response to P3 indicated that cyclization did not affect the critical TCR contact sites in MS PBMC. Interestingly, the cyclic P3 analog was found to be a stronger binder to HLA-DR4 compared to linear P2. Moreover, cyclic P3 was more stable to proteolysis compared to linear P2. Finally, both P2 and P3 suppressed EAE induced by an encephalitogenic guinea pig MBP74-85 epitope in Lewis rats whereas P1 failed to do so. In conclusion, cyclization of myelin altered peptide ligand (Ala91,Ala96)MBP87-99 improved binding affinity to HLA-DR4, resistance to proteolysis and antigen-specific immunomodulation, rendering cyclo(87-99)(Ala91,Ala96)MBP87-99 an important candidate drug for MS immunotherapy. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
    Full-text · Article · Jun 2015 · European Journal of Medicinal Chemistry
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    ABSTRACT: The conjugation of polysaccharides to peptides is essential for antigen delivery and vaccine development. Herein, we show that tricine SDS-PAGE in combination with Coomassie blue staining was adequate to determine the conjugation efficacy of a peptide (epitope 35-55 of myelin oligodendrocyte glycoprotein) to mannan. In addition, tricine SDS-PAGE and periodic acid-Schiff stains were able to monitor the redox state of mannan. Using the described protocol, more than 99.9% of a peptide containing five lysines at its N-terminus was confirmed conjugated to mannan. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Jun 2015 · Analytical Biochemistry
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    ABSTRACT: Peptides and proteins are attractive initial leads for the rational design of bioactive molecules. Several natural cyclic peptides have recently emerged as templates for drug design due to their resistance to chemical or enzymatic hydrolysis and high selectivity to receptors. The development of practical protocols that mimic the power of nature's strategies remains paramount for the advancement of novel peptide-based drugs. The de novo design of peptide mimetics (non-peptide molecules or cyclic peptides) for the synthesis of linear or cyclic peptides has enhanced the progress of therapeutics and diverse areas of science and technology. In the case of metabolically unstable peptide ligands, the rational design and synthesis of cyclic peptide analogues has turned into an alternative approach for improved biological activity. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · May 2015 · Biopolymers
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    ABSTRACT: Multiple sclerosis (MS) is a serious autoimmune demyelinating disease leading to loss of neurological function. The design and synthesis of various altered peptide ligands (APLs) of immunodominant epitopes of myelin proteins to alter the autoimmune response, is a promising therapeutic approach for MS. In this study, linear and cyclic peptide analogues based on the myelin basic protein 83-99 (MBP83-99) immunodominant epitope conjugated to reduced mannan via the (KG)5 and KLH bridge respectively, were evaluated for their biological/immunological profiles in SJL/J mice. Of all the peptide analogues tested, linear MBP83-99(F91) and linear MBP83-99(Y91) conjugated to reduced mannan via a (KG)5 linker and cyclic MBP83-99(F91) conjugated to reduce mannan via KLH linker, yielded the best immunological profile and constitute novel candidates for further immunotherapeutic studies against MS in animal models and in human clinical trials.
    Full-text · Article · Apr 2015 · Frontiers in Immunology
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    ABSTRACT: Background: Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose-response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed. Results: In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified β-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly. Conclusions: The study of a LHRH analogue conjugated with modified β-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery. General significance: NMR in combination with MD simulation is of great value for a successful rational design of peptide-cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.
    Full-text · Article · Jan 2015 · Biochimica et Biophysica Acta (BBA) - General Subjects
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    ABSTRACT: Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.
    Preview · Article · Nov 2014 · Molecules
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    ABSTRACT: Antigen presenting cells are critical for regulating immune responses. We tested mannan-peptide conjugates for targeting myelin peptides to APC to induce T cell tolerance and resistance to experimental autoimmune encephalomyelitis (EAE). Myelin peptides conjugated to mannan in oxidized (OM) or reduced (RM) forms protected mice against EAE in prophylactic and therapeutic protocols, with OM-conjugated peptides giving best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation, but not alterations in Th1, Th17 or Treg cell differentiation or T cell apoptosis compared to EAE controls. OM-MOG-loaded bone marrow-derived DC showed up-regulated expression of co-stimulatory molecules, reduced PD-L1 expression and enhanced CD40-inducible IL-12 and IL-23 production, features consistent with immunogenic DC. OM-MOG induced active T cell tolerance because i.d. administration or passive transfer of OM-MOG-loaded DC suppressed ongoing EAE, while OM-MOG-vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. As in vivo, MOG-specific T cells cultured with OM-MOG-loaded DC showed reduced proliferation and equal Th1 and Th17 cell differentiation as those with MOG-loaded DC, but surprisingly cytokine production was unresponsive to CD40 engagement. Impaired effector T cell function was further evidenced in spinal cord sections from OM-MOG-vaccinated EAE mice, where markedly reduced numbers of CD3(+) T cells were present, restricted to leptomeninges and exceptional parenchymal lesions. Our results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and APC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells. Copyright © 2014 Elsevier Inc. All rights reserved.
    No preview · Article · Oct 2014 · Experimental Neurology
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    ABSTRACT: ΑΤ1 receptor (AT1R) antagonists exert their antihypertensive effects by preventing the vasoconstrictive hormone AngII to bind to the AT1 receptor. It has been proposed that these biological effects are mediated through a two-step mechanism reaction. In the first step, they are incorporated in the core of the lipid bilayers and in the second step they reach the active site of the receptor through lateral diffusion. In this model, drug:membrane interactions are key elements for the drugs achieving inhibition at the AT1 receptor. In this work, the interactions of the prodrug candesartan cilexetil (TCV-116) with lipid bilayers are studied at molecular detail. Solid-state (13)C-CP/MAS, 2D (1)H-(1)H NOESY NMR spectroscopy and in silico calculations are used. TCV-116 and olmesartan, another drug which acts as an AT1R antagonist are compared for their dynamic effects in lipid bilayers using solid-state (2)H-NMR. We find a similar localization of TCV-116 compared to other AT1 antagonists in the intermediate polar region. In addition, we can identify specific local interactions. These interactions may be associated in part with the discrete pharmacological profiles observed for different antagonists.
    Full-text · Article · Jun 2014 · Biochimica et Biophysica Acta (BBA) - Biomembranes
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    ABSTRACT: Background: The predominant proteins of the CNS are myelin basic protein, proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein. PLP139-151 is one of the major encephalitogenic epitopes of PLP. The epitope PLP139-151 binds to MHC class II (I-A(s)) of SJL/J mice and induces Th1 responses. Aim: The aim was to synthesize and test the immunological activity and cyclic analogs of PLP139-151 peptide and determine the immunological differences between adjuvant and conjugation to mannan. Materials & methods: We designed and synthesized cyclic peptides based on the linear PLP139-151 epitope by mutating critical T-cell receptor contact sites of residues W(144) and H(147), resulting in the mutant peptides PLP139-151, [L(144), R(147)]PLP139-151 or cyclo(139-151)PLP139-151 and cyclo(139-151) [L(144), R(147)]PLP139-151. In this study, mice were immunized with mutant peptides either emulsified in complete Freund's adjuvant or conjugated to reduced mannan and responses were assessed. Results: Linear double-mutant peptide [L(144), R(147)]PLP139-151 induced high levels of IL-4 responses and low levels of IgG total, and cyclization of this analog elicited low levels of IFN-γ. Moreover, linear [L(144), R(147)]PLP139-151 conjugated to reduced mannan did not induce IFN-γ, whilst both linear agonist PLP139-151 and cyclic agonist cyclo(139-151)PLP139-151 induced IFN-γ-secreting T cells. Molecular dynamics simulations of linear and cyclic(139-151)PLP139-151 analogs indicated the difference in topology of the most important for biological activity amino acids. Conclusion: Cyclic double-mutant analog cyclo(139-151) [L(144), R(147)]PLP139-151 has potential for further studies for the immunotherapy of multiple sclerosis.
    No preview · Article · Jun 2014 · Immunotherapy
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    ABSTRACT: The dissolution of the antihypertensive AT1 antagonist olmesartan in methanol generates in situ a new highly bioactive methyl ether analogue via SN1 mechanism involving an intramolecular proton transfer from carboxyl to hydroxyl group. Theoretical calculations confirmed the thermodynamic control preference of methyl ether versus the antagonistic product methyl ester. Α facile synthetic method for olmesartan methyl ether from olmesartan or olmesartan medoxomil is also described. Interestingly, the introduction of the methyl group to olmesartan did not alter its pharmacological properties. This observation opens new avenues in the synthesis of novel drugs, since hydroxyl and carboxylate groups have an orthogonal relationship in many drugs.
    No preview · Article · May 2014 · Combinatorial Chemistry & High Throughput Screening
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    Full-text · Dataset · Oct 2013
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    ABSTRACT: The ligand binding determinants for the Angiotensin II type 1 receptor (AT1R), a G protein-coupled receptor (GPCR), have been characterized by means of computer simulations. As a first step, a pharmacophore model of known AT1R ligands exhibiting a wide range of binding affinities was generated. Secondly, a structural model of AT1R was built making use of the growing set of crystal structures of GPCRs, which was further used for the docking of the AT1R ligands based on the devised pharmacophore model. Next, ligand - receptor - lipid bilayer systems were studied by means of molecular dynamics (MD) simulations. Overall, combining the pharmacophore model with binding free energy calculations obtained from the MD simulations, the present study has permitted to propose the molecular mechanisms by which sartans interact with AT1R.
    Full-text · Article · Oct 2013 · Journal of Chemical Information and Modeling
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    ABSTRACT: Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83-99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.
    Full-text · Article · Aug 2013 · Bioorganic & medicinal chemistry
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    ABSTRACT: In the present work, a facile and efficient route for the synthesis of a series of N-substituted imidazole derivatives is described. Docking studies have revealed that N-substituted imidazole derivatives based on (E)-urocanic acid may be potential antihypertensive leads. Therefore, new AT1 receptor blockers bearing either the benzyl or the biphenylmethyl moiety at the N-1 or N-3 position, either the (E)-acrylate or the propanoate fragment and their related acids at the C-4 position as well as a halogen atom at the C-5 position of the imidazole ring, were synthesized. The newly synthesized analogues were evaluated for binding to human AT1 receptor. The biological results showed that this class of molecules possesses moderate or no activity, thus not always confirming high docking scores. Nonetheless, important conclusions can be derived for their molecular basis of their mode of action and help medicinal chemists to design and synthesize more potent ones. An aliphatic group as in losartan seems to be important for enhancing binding affinity and activity.
    Full-text · Article · Jul 2013 · Molecules
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    ABSTRACT: This article describes the rational design, synthesis and pharmacological properties of amide-linked cyclic analogues of Luteinizing Hormone-Releasing Hormone (LHRH) with substitutions at positions 1 (Pro), 6 (d-Leu/d-Trp), 9 (Aze) and 10 (BABA/Acp). These LHRH analogues fulfil the conformational requirements that are known in the literature (bend in the 5-8 segment) to be essential for receptor recognition and activation. Although, they are characterised by an overall low binding affinity to the LHRH-I receptor, the cyclic analogues that were studied and especially the cyclo(1-10)[Pro(1), d-Leu(6), BABA(10)] LHRH, exhibit a profoundly enhanced in vitro and in vivo stability and improved pharmacokinetics in comparison with their linear counterpart and leuprolide. Upon receptor binding, cyclo(1-10)[Pro(1), d-Leu(6), BABA(10)] LHRH causes testosterone release in C57/B16 mice (in vivo efficacy) that is comparable to that of leuprolide. Testosterone release is an acutely dose dependent effect that is blocked by the LHRH-I receptor antagonist, cetrorelix. The pharmacokinetic advantages and efficacy of cyclo(1-10)[Pro(1), d-Leu(6), BABA(10)] LHRH render this analogue a promising platform for future rational drug design studies towards the development of non-peptide LHRH mimetics.
    Full-text · Article · Oct 2012 · European Journal of Medicinal Chemistry
  • Theodore V. Tselios · John M. Matsoukas

    No preview · Article · Sep 2012 · ChemInform
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    ABSTRACT: Introduction Gonadotropin-releasing hormone (GnRH) receptor agonists have wide clinical applications (prostate cancer, endocrine disorders) on the basis of their superiority, when compared to the native hormone (GnRH) in terms of potency (high receptor affinity) and improved proteolytic stability. However, GnRH analogues are still susceptible to the action of proteolytic enzymes and have limited absorption and low bioavailability. As a consequence, these peptides are administered intramuscularly or subcutaneously and as depot formulations compromising efficacy (being dependent on patient compliance). Therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. Taking into account the importance of hormonal therapy for the treatment of prostate cancer and endocrine disorders as well as the current needs for improved therapeutic approaches, we focused on efforts to discover pharmacokinetically superior and possibly equipotent novel GnRH peptide analogues. Although superagonists have well-established clinical benefits, it is true that their in vivo stability remains a limiting factor that most likely prevents them from exerting any direct effects on tumors (extrapituitary) or causing rapid desensitization of the GnRH-I receptor. Herein, we report our findings regarding analogue 1, [Des- Gly10,Tyr5(OMe),D-Leu6,Aze-NHEt9]GnRH, a molecule with the conformational features required for agonistic activity and enhanced in vitro and in vivo stability compared with leuprolide. Results and Discussion In vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatography-tandem mass spectrometry. Analogue 1, an agonist of the GnRH-I receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. Repeated dosing studies in mice demonstrated that analogue 1 was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. Analogue 1 also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (LNCaP) cells. Although analogue 1 has reduced binding affinity on the GnRH-I receptor compared with leuprolide, repeated dosing studies in mice demonstrated that analogue 1 was well tolerated and had potency similar to that of leuprolide. Analogue 1 was also endowed with significant anti-proliferative activity on prostate cancer cells (LNCaP), similar to that exerted by leuprolide (drug in the clinic). On the basis of its pharmacokinetic advantages, it is likely that the peptide analogue in question or analogues based on this new design will be therapeutically advantageous for the treatment of endocrine disorders or prostate cancer. Such pharmacokinetic advantages can be particularly valuable with respect to local/direct extrapituitary effects that will be essential for more effective treatment of cancer. References [1] Katsila, T.; Balafas, E.; Liapakis, G.; Limonta, P.; Montagnani-Marelli, M.; Gkountelias, K.; Tselios, T.; Kostomitsopoulos, N.; Matsoukas, J.; Tamvakopoulos, C. J Pharmacol Exp Ther. 2011, 336, 613
    Full-text · Conference Paper · Sep 2012
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    ABSTRACT: Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP(1-11)) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP(1-11)[4A]) or a tyrosine residue (Ac-MBP(1-11)[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-A(u) was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln(3) residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-A(u) complex, has a different orientation in the mutated analogues especially in the Ac-MBP(1-11)[4A] peptide. In particular the side chain of Gln(3) is not solvent exposed as for the native Ac-MBP(1-11) and it is not available for interaction with the TCR.
    Full-text · Article · Nov 2011 · Journal of Computer-Aided Molecular Design
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    ABSTRACT: The two new synthetic analogues of the MBP(83-99) epitope substituted at Lys(91) (primary TCR contact) with Phe [MBP(83-99) (Phe(91))] or Tyr [MBP(83-99) (Tyr(91))], 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 MBP(83-99) 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 MBP(83-99) 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.
    Full-text · Article · Sep 2011 · Journal of Computer-Aided Molecular Design

Publication Stats

795 Citations
197.00 Total Impact Points

Institutions

  • 1998-2015
    • University of Patras
      • • Department of Chemistry
      • • Division of Organic Chemistry Biochemistry and Natural Products
      • • School of Medicine
      Rhion, West Greece, Greece
  • 2008
    • University of Thessaly
      • Laboratory of Clinical Chemistry
      Iolcus, Thessaly, Greece
  • 2007
    • Burnet Institute
      • Centre for Immunology
      Melbourne, Victoria, Australia