Combined Computational and Structural Approach into Understanding the Role of Peptide Binding and Activation of Melanocortin Receptor 4.

Melanocortin receptor 4 (MC4R) is expressed predominantly in the central nervous system and regulates food intake and sexual function and is also thought to be responsible for effects on mood and cognition. It belongs to the melanocortin receptor subfamily of G protein-coupled receptors (GPCRs). Here, we have synthesized and structurally characterized three peptides that bind to MC4R, producing different signaling events. AgRP is a naturally occurring antagonist, HLWNRS is the minimal sequence of the N-terminal with partial agonist activity, and aMSH is a full agonistic peptide. By implementing molecular dynamics simulations on the different peptide-receptor complexes, we propose their molecular basis of binding to investigate their differential molecular properties regarding the activation states of the receptor. Our analysis shows that the agonist and partial agonist may induce rotation in transmembrane helix 3, which is known to be involved in the key events occurring during GPCR activation, and this movement is impacted by certain aromatic residues and their positioning in the orthosteric binding site of the receptor.

Myelin Oligodendrocyte Glycoprotein and Multiple Sclerosis.

BACKGROUND: Myelin oligodendrocyte glycoprotein (MOG) is located on the external surface of myelin, a membranous component of the central nervous system (CNS) that forms the insulating lipid layer around neurons. The major MOG splicing variant (a1 transcript) encodes a transmembrane protein with an extracellular domain of an Ig variable (IgV) fold. MOG IgV domains from the same or different cells dimerize and contribute to the organization and maintenance of the myelin sheath in neurons. The encepalitogenic T cells recognize MOG and its immunodominant epitopes (epitopes 1-22, 35-55 and 92-106 located at the dimer interface) as foreign antigens and cause the destruction of myelin (demyelination) leading to the clinical condition known as multiple sclerosis (MS). Recognition of the antigen takes place in the context of the trimolecular complex formed by HLA, MOGpeptides and TCR. OBJECTIVE: Understanding the role of MOG in MS. METHOD/RESULTS: We have reviewed herein, the genomic organization of the human MOG gene, the structural characteristics of the MOG protein, the involvement of MOG in MS and clinical studies for the treatment of MS based on MOG peptide analogues. CONCLUSION: Conjugates of antigenic MOG peptides to mannan and combinations of antigenic MOG and other peptides chemically linked to cells of the immune system may modify the immune response, alleviating in some cases the symptoms of MS.

Design, synthesis and evaluation of an anthraquinone derivative conjugated to myelin basic protein immunodominant (MBP85-99) epitope: Towards selective immunosuppression.

Anthraquinone type compounds, especially di-substituted amino alkylamino anthraquinones have been widely studied as immunosuppressants. The anthraquinone ring is part of mitoxandrone that has been used for the treatment of multiple sclerosis (MS) and several types of tumors. A desired approach for the treatment of MS would be the immunosuppression and elimination of specific T cells that are responsible for the induction of the disease. Herein, the development of a peptide compound bearing an anthraquinone derivative with the potential to specifically destroy the encephalitogenic T cells responsible for the onset of MS is described. The compound consists of the myelin basic protein (MBP) 85-99 immunodominant epitope (MBP85-99) coupled to an anthraquinone type molecule (AQ) via a disulfide (S-S) and 6 amino hexanoic acid (Ahx) residues (AQ-S-S-(Ahx)6MBP85-99). AQ-S-S-(Ahx)6MBP85-99 could bind to HLA II DRB1*-1501 antigen with reasonable affinity (IC50 of 56 nM) The compound was localized to the nucleus of Jurkat cells (an immortalized line of human T lymphocytes) 10 min after its addition to the medium and resulted in lowered Bcl-2 levels (apoptosis). Entrance of the compound was abolished when cells were pre-treated with cisplatin, an inhibitor of thioredoxin reductase. Accordingly, levels of free thiols were elevated in the culture supernatants of Jurkat cells exposed to N-succinimidyl 3-(2-pyridyldithio) propionate coupled to (Ahx)6MBP85-99 via a disulphide (SPDP-S-S-(Ahx)6MBP85-99) but returned to normal after exposure to cisplatin. These results raise the possibility of AQ-S-S-(Ahx)6MBP85-99 being used as an eliminator of encephalitogenic T cells via implication of the thioredoxin system for the generation of the toxic, thiol-containing moiety (AQ-SH). Future experiments would ideally determine whether SPDP-S-S-(Ahx)6MBP85-99 could incorporate into HLA II DRB1*-1501 tetramers and neutralize encephalitogenic T cell lines sensitized to MBP85-99.

Mannosylated Linear and Cyclic Single Amino Acid Mutant Peptides Using a Small 10 Amino Acid Linker Constitute Promising Candidates Against Multiple Sclerosis.

Multiple sclerosis (MS) is a serious autoimmune demyelinating disease leading to loss of neurological function. The design and synthesis of various altered peptide ligands 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 analogs based on the myelin basic protein 83-99 (MBP83-99) immunodominant epitope conjugated to reduced mannan via the (KG)5 and keyhole limpet hemocyanin (KLH) bridge, respectively, were evaluated for their biological/immunological profiles in SJL/J mice. Of all the peptide analogs tested, linear MBP83-99(F(91)) and linear MBP83-99(Y(91)) conjugated to reduced mannan via a (KG)5 linker and cyclic MBP83-99(F(91)) 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.

Conjugation of a peptide to mannan and its confirmation by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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.

Review cyclic peptides on a merry-go-round; towards drug design.

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 (nonpeptide 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.

Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

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.