RESUMO
We report the novel synthesis of cyclic PLP139-151 (cPLP) and its application in SJL/J mice to study its encephalitogenic effects. Our results indicate that the cPLP analog is minimally encephalitogenic when administered to induce experimental autoimmune encephalomyelitis (low disease burden, minimal inflammatory, demyelinating and axonopathic pathology compared to its linear counterpart). Proliferation assays confirmed the low stimulatory potential of the cPLP compared to linPLP (2.5-fold lower proliferation) as well as inducing lower antibody responses. Molecular modeling showed a completely different TCR recognition profile of cPLP in regard to linPLP, where H147 replaces W144 and F151-K150 replace H147 as TCR contacts, which may explain the difference on each peptide's response.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos Cíclicos/imunologia , Sequência de Aminoácidos , Animais , Ciclização , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Epitopos/efeitos adversos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Camundongos Endogâmicos , Simulação de Acoplamento Molecular , Proteína Proteolipídica de Mielina/efeitos adversos , Proteína Proteolipídica de Mielina/química , Proteína Proteolipídica de Mielina/metabolismo , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/efeitos adversos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Medula Espinal/patologia , Linfócitos T/metabolismoRESUMO
EAE is induced to susceptible mice using linear peptides of myelin proteins of the central nervous system. Specific peptide motifs within the peptide-binding groove of the MHC peptide-complex determines the affinity of the peptide in each animal and the consequent T-cell receptor recognition and activation of the cell. Altered peptide ligand (APL) vaccination is a novel approach based on an effort to induce T-cell tolerance or alter cytokine profile from pro-inflammatory to anti-inflammatory. In the present study we synthesized the MOG35-55 peptide and altered its 3-dimensional conformation to make it a cyclic one (c-MOG35-55). EAE was induced in C57BL/6 mice and pathology was studied on acute and chronic phase of the disease. Our data indicates that c-MOG35-55 peptide alone induces a mild transient acute phase without chronic axonopathy. Administration of the c-MOG35-55 peptide at a 1:1 ratio during disease induction significantly ameliorates clinical disease and underlying pathology, such as demyelination and axonopathy in the acute and chronic phases. Binding and structural studies revealed milder interactions between the c-MOG35-55 and mouse or human MHC class II alleles (H2-IAb and HLA-DR2). Collectively, we provide data supporting for the first time the concept that the cyclic modification of an established encephalitogenic peptide ameliorates the clinical outcomes and underlying pathological processes of EAE. Such a cyclic modification of linear peptides could provide a novel treatment approach for future, patient-selective, immunomodulative treatments of multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Peptídeos Cíclicos/uso terapêutico , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Amino acid mutations to agonist peptide epitopes of myelin proteins have been used to modulate immune responses and experimental autoimmune encephalomyelitis (EAE, animal model of multiple sclerosis). Such amino acid alteration are termed, altered peptide ligands (APL). We have shown that the agonist myelin basic protein (MBP) 87-99 epitope (MBP87-99) with crucial T cell receptor (TCR) substitutions at positions 91 and 96 (K91,P96 (TCR contact residues) to R91,A96; [R91,A96]MBP87-99) results in altered T cell responses and inhibits EAE symptoms. In this study, the role of citrullination of arginines in [R91,A96]MBP87-99 peptide analog was determined using in vivo experiments in combination with computational studies. The immunogenicity of linear [Cit91,A96,Cit97]MBP87-99 and its cyclic analog - cyclo(87-99)[Cit91,A96,Cit97]MBP87-99 when conjugated to the carrier mannan (polysaccharide) were studied in SJL/J mice. It was found that mannosylated cyclo(87-99)[Cit91,A96,Cit97]MBP87-99 peptide induced strong T cell proliferative responses and IFN-gamma cytokine secretion compared with the linear one. Moreover, the interaction of linear and cyclic peptide analogs with the major histocompatibility complex (MHC II, H2-IAs) and TCR was analyzed using molecular dynamics simulations at the receptor level, in order to gain a better understanding of the molecular recognition mechanisms that underly the different immunological profiles of citrullinated peptides compared to its agonist native counterpart MBP87-99 epitope. The results demonstrate that the citrullination of arginine in combination with the backbone conformation of mutated linear and cyclic analogs are significant elements for the immune response triggering the induction of pro-inflammatory cytokines.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Animais , Proliferação de Células , Citocinas/imunologia , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/imunologia , Camundongos , Camundongos Endogâmicos , Simulação de Dinâmica Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Linfócitos T/citologiaRESUMO
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) of autoimmune etiology that results from an imbalance between CNS-specific T effector cells and peripheral suppressive mechanisms mediated by regulatory cells (RC). In this research, we collected blood samples from 83 relapsing remitting MS (RRMS) patients and 45 healthy persons (HC), to assess the sizes of their RC populations, including CD4âºCD25(high)Foxp3⺠(nTregs), CD3âºCD4âºHLA(-)Gâº, CD3âºCD8âºCD28(-), CD3âºCD56âº, and CD56(bright) cells, and how RC are affected by disease activity (acute phase or remission) and types of treatment (methylprednisolone, interferon, or natalizumab). In addition, we isolated peripheral blood mononuclear cells (PBMC) and cultured them with peptides mapping to myelin antigens, to determine RC responsiveness to autoantigens. The results showed decreased levels of nTregs in patients in the acute phase ± methylprednisolone and in remission + natalizumab, but HC levels in patients in remission or receiving interferon. Patients + interferon had the highest levels of CD3âºCD4âºHLA(-)G⺠and CD3âºCD8âºCD28(-) RC, and patients in the acute phase + methylprednisolone the lowest. Patients in remission had the highest levels of CD3âºCD56âº, and patients in remission + natalizumab the highest levels of CD56(bright) cells. Only nTregs responded to autoantigens in culture, regardless of disease activity or treatment. The highest suppressive activity was exhibited by nTregs from patients in remission. In conclusion, in RRMS disease activity and type of treatment affect different RC populations. nTregs respond to myelin antigens, indicating that it is possible to restore immunological tolerance through nTreg induction.
Assuntos
Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/patologia , Linfócitos T Reguladores/imunologia , Adulto , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Proteína Básica da Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Natalizumab/uso terapêuticoRESUMO
There is a need in autoimmune diseases to uncover the mechanisms involved in the natural resolution of inflammation. In this article, we demonstrate that granulocytic myeloid-derived suppressor cells (G-MDSCs) abundantly accumulate within the peripheral lymphoid compartments and target organs of mice with experimental autoimmune encephalomyelitis prior to disease remission. In vivo transfer of G-MDSCs ameliorated experimental autoimmune encephalomyelitis, significantly decreased demyelination, and delayed disease onset through inhibition of encephalitogenic Th1 and Th17 immune responses. Exposure of G-MDSCs to the autoimmune milieu led to up-regulation of the programmed death 1 ligand that was required for the G-MDSC-mediated suppressive function both in vitro and in vivo. Importantly, myeloid-derived suppressor cells were enriched in the periphery of subjects with active multiple sclerosis and suppressed the activation and proliferation of autologous CD4(+) T cells ex vivo. Collectively, this study revealed a pivotal role for myeloid-derived suppressor cells in the regulation of multiple sclerosis, which could be exploited for therapeutic purposes.
Assuntos
Doenças Autoimunes/imunologia , Doenças do Sistema Nervoso Central/imunologia , Granulócitos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Transplante de Células , Doenças Desmielinizantes/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Camundongos , Esclerose Múltipla/imunologia , Células Mieloides/imunologiaRESUMO
BACKGROUND: Methamphetamine (METH) substance-use disorder is an ever-growing global health issue with no effective treatment. Anti-METH vaccines are under investigation as an alternative to existing psychological interventions. This platform has made significant progress over past decades mainly in preclinical stages, and efforts to develop an anti-METH vaccine with a high antibody response are of utmost importance. METHODOLOGY: A novel conjugated anti-METH vaccine was developed using METH HCl as the starting material for the design of hapten, a peptide linker consisting of five lysines and five glycines, and finally immunogenic carrier mannan, which is novel to this platform. All the chemical reaction steps were confirmed by several analytical techniques, and the immunogenicity of the developed vaccine was investigated in a mouse model. RESULTS: Thin-layer chromatography and gas chromatography confirmed the reaction between METH and peptide linker. UV, NMR and color tests were used to confirm the presence of the aldehyde groups in oxidized mannan (OM). The final conjugated vaccine was confirmed by UV and LC-MS. The stability of mannan, the METH hapten, and the final vaccine was evaluated by UV and LC-MS and demonstrated satisfactory stability over 3 months in various storage conditions. Animal studies supported the immunogenicity of the novel vaccine. CONCLUSIONS: We successfully developed and characterized a novel METH vaccine in vitro and in vivo. The present study findings are encouraging and will form the basis of further exploration to assess its effectiveness to prevent METH addiction in preclinical models.
RESUMO
BACKGROUND: Methamphetamine (METH) addiction is a major public health concern globally with limited management options. The development of a METH vaccine through hapten design has received significant attention as a promising platform for the potential treatment of METH addiction and overdose, however there is yet to be a successful candidate in human trials. RESEARCH DESIGN AND METHODS: In this study, we developed a novel conjugated METH vaccine using oxidized mannan (a polymannose) as an immunogenic carrier. A METH hapten was synthesized by using amphetamine and conjugated to mannan with a (Lysine-Glycine-Lysine-Glycine-lysine-Glycine-Lysine-Glycine-Lysine-Glycine) (KG)5 peptide linker. RESULTS: The reaction between amphetamine and (KG)5, oxidation of mannan, and conjugation of amphetamine-(KG)5 with oxidized mannan were confirmed by color tests, Fourier-transform infrared spectroscopy, gas and liquid chromatography mass spectrometry, thin-layer chromatography, and ultraviolet spectrophotometer. Additionally, the ability of the vaccine to generate antibodies was confirmed in C57BL/6 mice. CONCLUSIONS: The successful development and characterization of the METH-mannan conjugate vaccine, provides a potential therapeutic intervention to curb METH substance use disorders. Each step of vaccine development was characterized to aid in future research on these vaccines, and the immunogenicity shown in the animal models supports future evaluation of the approach. Future studies of the conjugated METH vaccine should evaluate the efficacy in animal models of acute and chronic METH to pave the way for human studies.
Assuntos
Transtornos Relacionados ao Uso de Anfetaminas , Metanfetamina , Transtornos Relacionados ao Uso de Anfetaminas/tratamento farmacológico , Transtornos Relacionados ao Uso de Anfetaminas/prevenção & controle , Animais , Glicina/uso terapêutico , Haptenos , Humanos , Lisina/uso terapêutico , Mananas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Vacinas ConjugadasRESUMO
Myelin peptide-mannan conjugates have been shown to be potential vaccines in the immunotherapy of multiple sclerosis. The conjugates are comprised from the epitope peptide and the polysaccharide mannan which transfers as a carrier the antigenic peptide to dendritic cells that process and present antigenic peptides at their surface in complex with MHC class I or class II resulting in T-cell stimulation. The conjugation of antigenic peptide with mannan occurs through the linker (Lys-Gly)5, which connects the peptide with the oxidized mannose units of mannan. This study describes novel methods for the quantification of the vaccine ingredient peptide within the conjugate, a prerequisite for approval of clinical trials in the pursuit of multiple sclerosis therapeutics. Myelin peptides, such as MOG35-55, MBP83-99, and PLP131-145 in linear or cyclic form, as altered peptide ligands or conjugated to appropriate carriers, possess immunomodulatory properties in experimental models and are potential candidates for clinical trials.
RESUMO
Myelin basic protein peptide 83-99 (MBP83-99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP(83-99) has been performed using 2D (1)H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined alpha-helix spanning residues Val(87)-Phe(90) is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.
Assuntos
Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/química , Proteína Básica da Mielina/imunologia , Proteínas do Tecido Nervoso/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fatores de Transcrição/química , Algoritmos , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Dimetil Sulfóxido/química , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Proteína Básica da Mielina/síntese química , Fragmentos de Peptídeos/síntese química , Conformação Proteica , Software , Solventes/química , Propriedades de SuperfícieRESUMO
Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF(1)), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF(1) we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF(1). The W259A, F260A, and W259A/F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF(1) compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF(1). This is the first time that a specific region of CRF(1) has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.
Assuntos
Alanina/genética , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Ligação Competitiva/genética , Linhagem Celular , Humanos , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/química , Hormônios Peptídicos/química , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/genéticaRESUMO
Mutations of peptides to generate altered peptide ligands, capable of switching immune responses from T helper 1 (Th1) to T helper 2 (Th2), are promising candidates for the immunotherapy of autoimmune diseases such as multiple sclerosis (MS). We synthesized two mutant peptides from myelin basic protein 87-99 (MBP(87-99)), an immunodominant peptide epitope identified in MS. Mutations of residues K(91) and P(96), known to be critical T-cell receptor (TCR) contact sites, resulted in the mutant peptides [R(91), A(96)]MBP(87-99) and [A(91), A(96)]MBP(87-99). Immunization of mice with these altered peptide ligands emulsified in complete Freund's adjuvant induced both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) responses compared with only IFN-gamma responses induced to the native MBP(87-99) peptide. It was of interest that [R(91), A(96)]MBP(87-99) conjugated to reduced mannan induced 70% less IFN-gamma compared with the native MBP(87-99) peptide. However, [A(91), A(96)]MBP(87-99) conjugated to reduced mannan did not induce IFN-gamma-secreting T cells, but elicited very high levels of interleukin-4 (IL-4). Furthermore, antibodies generated to [A(91), A(96)]MBP(87-99) peptide conjugated to reduced mannan did not cross-react with the native MBP(87-99) peptide. By molecular modelling of the mutant peptides in complex with major histocompatibility complex (MHC) class II, I-A(s), novel interactions were noted. It is clear that the double-mutant peptide analogue [A(91), A(96)]MBP(87-99) conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies investigating potential treatments for MS.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Mananas/imunologia , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Animais , Efeito Espectador/imunologia , Reações Cruzadas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Adjuvante de Freund , Imunização/métodos , Imunoglobulina G/biossíntese , Interferon gama/biossíntese , Interleucina-4/biossíntese , Ligantes , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Mutação , Proteína Básica da Mielina/genética , Fragmentos de Peptídeos/genética , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Multiple sclerosis (MS) is an autoimmune demyelinating disease mediated primarily by CD4+ T cells. The design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of MS. We designed and synthesized a number of peptide analogs by mutating the principal TCR contact residue based on MBP83-99 epitope and these peptides were conjugated to reduced mannan. Immune responses were diverted from Th1 to Th2 in SJL/J mice and generated antibodies which did not cross-react with native MBP protein. Peptide [Y91]MBP83-99 gave the best cytokine and antibody profile and constitutes a promising candidate peptide for immunotherapy of MS. Structural alignment of existing crystal structures revealed the peptide binding motif of I-As. Molecular modeling was used to identify H-bonding and van der Waals interactions between peptides and MHC (I-A(s)).
Assuntos
Ativação Linfocitária/imunologia , Manose/metabolismo , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Processamento de Proteína Pós-Traducional/fisiologia , Células Th1/imunologia , Células Th2/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Células Cultivadas , Reações Cruzadas/imunologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Mananas/imunologia , Manose/imunologia , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismoRESUMO
A number of treatment options are available to multiple sclerosis patients, however this needs to be improved. Herein, we designed and synthesized a number of peptides by mutating principal TCR contact residues based on MBP(83-99) peptide epitope. Immunization of SJL/J mice with MBP(83-99) and mutant [A(91)]MBP(83-99), [E(91)]MBP(83-99), [F(91)]MBP(83-99), [Y(91)]MBP(83-99), and [R(91), A(96)]MBP(83-99) peptides, induced IFN-gamma, and only [R(91), A(96)]MBP(83-99) mutant peptide was able to induce IL-4 secretion by T cells. T cells against the native MBP(83-99) peptide cross-reacted with all peptides except [Y(91)]MBP(83-99) and [R(91),A(96)]MBP(83-99). The double mutant [R(91), A(96)]MBP(83-99) was able to antagonize IFN-gamma production in vitro by T cells against the native MBP(83-99) peptide. Antibodies generated to [R(91), A(96)]MBP(83-99) did not cross-react with whole MBP protein. Molecular modeling between peptide analogs and H2 I-A(s) demonstrated novel interactions. The [R(91), A(96)]MBP(83-99) double mutant peptide analog is the most promising for further therapeutic studies.
Assuntos
Interferon gama/metabolismo , Interleucina-4/metabolismo , Mutação/fisiologia , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/farmacologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Modelos Moleculares , Proteína Básica da Mielina/química , Fragmentos de Peptídeos/química , Linfócitos T/metabolismo , Fatores de TempoRESUMO
Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. Numerous treatment options are available to MS patients; however, these options need to be improved. Herein, we review the current drugs and therapeutic approaches available to MS patients, preclinical trial interventions and recent animal model studies for the potential therapy of MS. Since the current treatment of MS remains elusive and is limited, animal studies and clinical research offers an optimistic outlook.
Assuntos
Anti-Inflamatórios/uso terapêutico , Fatores Imunológicos/uso terapêutico , Imunossupressores/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/prevenção & controle , Peptídeos/uso terapêutico , Vacinas/uso terapêutico , Animais , Anti-Inflamatórios/imunologia , Humanos , Fatores Imunológicos/imunologia , Imunossupressores/imunologia , Esclerose Múltipla/imunologia , Peptídeos/imunologia , Vacinas/imunologiaRESUMO
BACKGROUND: Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system. MS is a T cell-mediated disease characterized by the proliferation, infiltration, and attack of the myelin sheath by immune cells. Previous studies have shown that cyclization provides molecules with strict conformation that could modulate the immune system. METHODS: In this study, we synthesized peptide analogues derived from the myelin basic protein (MBP)82â»98 encephalitogenic sequence (dirucotide), the linear altered peptide ligand MBP82â»98 (Ala91), and their cyclic counterparts. RESULTS: The synthesized peptides were evaluated for their binding to human leukocyte antigen (HLA)-DR2 and HLA-DR4 alleles, with cyclic MBP82â»98 being a strong binder with the HLA-DR2 allele and having lower affinity binding to the HLA-DR4 allele. In a further step, conformational analyses were performed using NMR spectroscopy in solution to describe the conformational space occupied by the functional amino acids of both linear and cyclic peptide analogues. This structural data, in combination with crystallographic data, were used to study the molecular basis of their interaction with HLA-DR2 and HLA-DR4 alleles. CONCLUSION: The cyclic and APL analogues of dirucotide are promising leads that should be further evaluated for their ability to alter T cell responses for therapeutic benefit against MS.
RESUMO
There is a need for novel drugs for the treatment of infectious diseases, autoimmunity and cancer. Cyclic peptides constitute a class of compounds that have made crucial contributions to the treatment of certain diseases. Penicillin, Vancomycin, Cyclosporin, the Echinocandins and Bleomycin are well-known cyclic peptides. Cyclic peptides, compared to linear peptides, have been considered to have greater potential as therapeutic agents due to their increased chemical and enzymatic stability, receptor selectively, and improved pharmacodynamic properties. They have been used as synthetic immunogens, transmembrane ion channels, antigens for Herpes Simplex Virus, potential immunotherapeutic vaccines for diabetes and Experimental Autoimmune Encephalomyelitis - an animal model of Multiple Sclerosis, as inhibitors against alpha-amylase and as protein stabilizers. Herein, we review important cyclic peptides as therapeutic agents in disease.
Assuntos
Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/uso terapêutico , Sequência de Aminoácidos , Produtos Biológicos , Cisteína , Dissulfetos/química , Humanos , Modelos Moleculares , Conformação Proteica , Vacinas SintéticasRESUMO
This work proposes a structural motif for the inhibition of experimental autoimmune encephalomyelitis (EAE) by the linear altered peptide ligands (APLs) [Ala91,96] MBP87-99 and [Arg91,Ala96] MBP87-99 of myelin basic protein. Molecular dynamics was applied to reveal distinct populations of EAE antagonist [Ala91,96] MBP87-99 in solution, in agreement with NOE data. The combination of the theoretical and experimental results led to the identification of a putative active conformation. This approach is of value as no crystallographic data is available for the APL-receptor complex. TCR contact residue Phe89 has an altered topology in the putative bioactive conformations of both APLs with respect to the native peptide, as found via crystallography; it is no longer prominent and solvent exposed. It is proposed that the antagonistic activity of the APLs is due to their binding to MHC, preventing the binding of self-myelin epitopes, with the absence of an immunologic response as the loss of some interactions with the TCR hinders activation of T-cells.
Assuntos
Modelos Moleculares , Proteína Básica da Mielina/química , Fragmentos de Peptídeos/química , Receptores de Antígenos de Linfócitos T/agonistas , Epitopos , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Soluções , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Experimental allergic encephalomyelitis (EAE) is a T helper 1 (Th1) mediated autoimmune disease and the principal animal model for multiple sclerosis (MS). Like MS, EAE is characterized by a coordinated inflammatory attack on the myelin sheath in the central nervous system (CNS), with damage to axons. No matter whether the ideal animal model is not yet available, much knowledge concerning the pathogenesis of MS has been achieved through studies on EAE. Dissecting the underlying immune mechanisms provided recognition of several myelin antigens that are vulnerable in autoimmune attack. The beneficial effect and the mechanism of action of a number of the currently used immunomodulating agents in MS therapy were first indicated in EAE. Altered peptide ligands (APL) can modulate T-cell responses to native peptide antigens implicated in the pathogenesis of autoimmune diseases such as MS and EAE. However, peptide therapy is hindered due to the sensitivity of peptides to proteolytic enzymes as well as due to some immune-mediated side effects. A number of cyclic myelin peptide analogs seem to be potential candidates in maintaining the biological function of the original peptide and effective in controlling inflammation in EAE. Additional data regarding the immunomodulating and neuroprotective effect of these much promising agents is required. Based on the data from studies on EAE models, clinical trials should also be designed in order to elucidate the impact of such APL-induced immune responses in MS disease activity. These clinical trials should carefully incorporate monitoring of both clinical, neuroimaging and immunological parameters.
Assuntos
Doenças Autoimunes/terapia , Doenças do Sistema Nervoso Central/terapia , Imunossupressores/uso terapêutico , Imunoterapia , Peptídeos/uso terapêutico , Animais , Doenças Autoimunes/imunologia , Biomimética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/terapia , Inflamação/terapia , Esclerose Múltipla/terapia , Proteínas da Mielina/uso terapêuticoRESUMO
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.
Assuntos
Imunoterapia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/farmacologia , Fragmentos de Peptídeos/farmacologia , Adolescente , Adulto , Idoso , Animais , Proliferação de Células/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Ligantes , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Esclerose Múltipla/patologia , Proteína Básica da Mielina/síntese química , Proteína Básica da Mielina/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Ratos , Ratos Endogâmicos Lew , Adulto JovemRESUMO
Protein interactions within regulatory networks should adapt in a spatiotemporal-dependent dynamic environment, in order to process and respond to diverse and versatile cellular signals. However, the principles governing recognition pliability in protein complexes are not well understood. We have investigated a region of the intrinsically disordered protein myelin basic protein (MBP(145-165)) that interacts with calmodulin, but that also promiscuously binds other biomolecules (membranes, modifying enzymes). To characterize this interaction, we implemented an NMR spectroscopic approach that calculates, for each conformation of the complex, the maximum occurrence based on recorded pseudocontact shifts and residual dipolar couplings. We found that the MBP(145-165)-calmodulin interaction is characterized by structural heterogeneity. Quantitative comparative analysis indicated that distinct conformational landscapes of structural heterogeneity are sampled for different calmodulin-target complexes. Such structural heterogeneity in protein complexes could potentially explain the way that transient and promiscuous protein interactions are optimized and tuned in complex regulatory networks.