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1.
Pharmaceutics ; 14(9)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36145590

RESUMO

Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases, but requires the incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface-modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2 kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 µm diameter) surface-modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface-modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 µL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice with minimal lung inflammation. Thus, surface modification of ~1 µm biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.

2.
ACS Infect Dis ; 6(5): 1169-1181, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32233506

RESUMO

EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro7 and N-methyl-Leu8 (Me-Leu8) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro7 and Me-Leu8 and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine7 and/or leucine8) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.


Assuntos
Adjuvantes Imunológicos/química , Complemento C5a , Oligopeptídeos/química , Substituição de Aminoácidos , Humanos
3.
Neurobiol Dis ; 114: 45-52, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29409952

RESUMO

OBJECTIVE: Adenosine participates in maintaining the excitatory/inhibitory balance in neuronal circuits. Studies indicate that adenosine levels in the cortex and hippocampus increase and exert sleep pressure in sleep-deprived and control animals, whereas in epilepsy reduced adenosine tone promotes hyperexcitability. To date, the role of adenosine in pathological conditions that result in both seizures and sleep disorders is unknown. Here, we determined adenosine tone in sleep and seizure regulating brain regions of Kv1.1 knockout (KO) mice, a model of temporal epilepsy with comorbid sleep disorders. METHODS: 1) Reverse phase-high performance liquid chromatography (RP-HPLC) was performed on brain tissue to determine levels of adenosine and adenine nucleotides. 2) Multi-electrode array extracellular electrophysiology was used to determine adenosine tone in the hippocampal CA1 region and the lateral hypothalamus (LH). RESULTS: RP-HPLC indicated a non-significant decrease in adenosine (~50%, p = 0.23) in whole brain homogenates of KO mice. Regional examination of relative levels of adenine nucleotides indicated decreased ATP and increased AMP in the cortex and hippocampus and increased adenosine in cortical tissue. Using electrophysiological and pharmacological techniques, estimated adenosine levels were ~35% lower in the KO hippocampal CA1 region, and 1-2 fold higher in the KO LH. Moreover, the increased adenosine in KO LH contributed to lower spontaneous firing rates of putative wake-promoting orexin/hypocretin neurons. INTERPRETATION: This is the first study to demonstrate a direct correlation of regionally distinct dichotomous adenosine levels in a single model with both epilepsy and comorbid sleep disorders. The weaker inhibitory tone in the dorsal hippocampus is consistent with lower seizure threshold, whereas increased adenosine in the LH is consistent with chronic partial sleep deprivation. This work furthers our understanding of how adenosine may contribute to pathological conditions that underlie sleep disorders within the epileptic brain.


Assuntos
Adenosina/metabolismo , Modelos Animais de Doenças , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Transtornos do Sono-Vigília/metabolismo , Transtornos do Sono-Vigília/fisiopatologia , Animais , Comorbidade , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Técnicas de Cultura de Órgãos
4.
Synthesis (Stuttg) ; 46(4): 515-521, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29097824

RESUMO

An efficient two-step synthesis of 4(5)-benzyl-L-histidine from L-histidine was developed. A Pictet-Spengler reaction between L-histidine and benzaldehyde in the presence of excess strong base yielded 4-phenylspinacine within one hour. Catalytic transfer hydrogenolysis in methanol at reflux using ammonium formate rapidly converted 4-L-phenylspinacine to 4(5)-benzyl-L-histidine within five minutes. No racemization of the final product 4(5)-benzyl-L-histidine was observed using the Marfey reagent. To show the utility of this methodology, a series of fluorinated benzylhistidines is presented.

6.
Chem Biol Drug Des ; 70(3): 216-26, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17718716

RESUMO

Synthesis of the first irreversible calcitonin gene-related peptide receptor antagonists is described. bis-(2-chloroethyl)amino and fluorosulphonyl groups were incorporated into the 4-position of the N-terminal benzoyl group of a potent competitive antagonist, N-alpha-benzoyl-h-alpha-CGRP(8-37) (analogues 4 and 6). Based on previous structure-activity relationships, a second pair of N-terminally modified analogues was synthesized containing a novel benzylated-His residue in position 10 (analogues 5 and 7). In separate experiments, SK-N-MC cells and mouse thoracic aortas were bathed in solutions containing 5 microM and 1.5 microM of each analogue, respectively. After extensive washing, calcitonin gene-related peptide concentration-response curves were generated for cAMP production in SK-N-MC cells and relaxation of mouse aortas. All analogues caused >20% reductions in maximal calcitonin gene-related peptide efficacy in both assays with analogue 5 containing an N-terminal bis-(2-chloroethyl)amino-benzoyl group and a benzylated-His10 residue completely abolishing cAMP production in SK-N-MC cells. Reductions in maximal responses were dependent on the analogue concentration. Analogue 4 also caused more than 10-fold reductions in the potency of the calcitonin gene-related peptide-mediated effects, whereas analogues 5, 6 and 7 have no significant effect on calcitonin gene-related peptide potency. These data indicate that all analogues bind irreversibly to calcitonin gene-related peptide receptors. The bis-(2-chloroethyl)amino-modified analogues 4 and 5 were more effective than the fluorosulphonyl-modified analogues 6 and 7.


Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/análogos & derivados , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cloro/química , Flúor/química , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Enxofre/química , Aminação , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Humanos , Masculino , Camundongos , Estrutura Molecular
7.
J Pharmacol Exp Ther ; 319(2): 749-57, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16873605

RESUMO

Human alpha-calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that produces a variety of cardiovascular and other effects via activation of specific CGRP receptors that produce cAMP. Functional CGRP receptors are a heterodimeric complex composed of the heptahelical calcitonin receptor-like receptor and the single transmembrane receptor activity-modifying protein 1. Based on the known structures of the antagonist CGRP((8-37)) and the human CGRP receptor, we designed novel CGRP receptor peptide antagonists with modifications to promote high affinity and selectivity for human CGRP receptors. Antagonist affinity (K(B)) at CGRP receptors was determined using the mouse thoracic aorta and human SK-N-MC cells. In aorta, CGRP((8-37)), [N-alpha-benzoyl]human alpha-CGRP((8-37)) [bzl-CGRP((8-37))], and [N-alpha-benzoyl-His(10)-benzyl]human alpha-CGRP((8-37)) [bzl-bn-CGRP((8-37))] caused rightward shifts in the concentration-response relaxation curve for CGRP with K(B) values of 1000, 88, and 50 nM, respectively. In human SK-N-MC cells, CGRP((8-37)), bzl-CGRP((8-37)), and bzl-bn-CGRP((8-37)) caused rightward shifts in the concentration-response curve for CGRP-stimulated cAMP production with K(B) values of 797, 15, and 0.63 nM, respectively. Thus, CGRP((8-37)) had the same affinity for human and mouse CGRP receptors, whereas bzl-CGRP((8-37)) and bzl-bn-CGRP((8-37)) displayed 6- and 80-fold higher affinities, respectively, for human CGRP receptors. In addition, the selectivity of the antagonists for human CGRP receptors was highly correlated with the antagonist hydrophobicity index. These relatively high-affinity, species-selective peptide antagonists provide novel tools to differentiate structural and functional features that are unique to the human CGRP receptor. Thus, these analogs may be useful compounds for development of drugs to treat migraine headache and other cardiovascular diseases.


Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Camundongos , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/química , Especificidade da Espécie , Relação Estrutura-Atividade
8.
J Cell Sci ; 118(Pt 19): 4333-41, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16159957

RESUMO

In previous studies in Drosophila, Nielsen et al. hypothesized that the beta tubulin C-terminal axonemal motif ;EGEFXXX', where X is an acidic amino acid, is required for ciliary function and assembly (Nielsen et al., 2001, Curr. Biol. 11, 529-533). This motif is present in some but not all mammalian beta tubulin isotypes. We therefore investigated whether this motif is important in ciliary function in mammals. In a preparation of isolated, ATP-reactivated bovine tracheal cilia, we found that monoclonal antibodies directed against the C-terminus of betaI, betaIV and betaV tubulin blocked ciliary beating in a concentration dependent manner. Antibodies against other epitopes of beta tubulin were ineffective, as were antibodies against alpha tubulin. Peptides consisting of the axonemal motif and motif-like sequences of these isotypes blocked ciliary beating. These results suggest that the axonemal motif sequences of betaI, betaIV and betaV tubulin are essential for ciliary function. Peptides consisting of corresponding C-terminal sequences in alpha tubulin isotypes were also ineffective in blocking ciliary beating, which suggests that the C-terminus of alpha tubulin is not directly involved in cilia function in mammals.


Assuntos
Sequência de Aminoácidos , Cílios/metabolismo , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Anticorpos/metabolismo , Bovinos , Cílios/ultraestrutura , Epitopos , Microtúbulos/metabolismo , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Mucosa Respiratória/citologia , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
9.
Biochem Biophys Res Commun ; 320(1): 170-5, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15207717

RESUMO

The tailed frog Ascaphus truei occupies a unique position in phylogeny as the most primitive extant anuran and is regarded as the sister taxon to the clade of all other living frogs. Eight structurally related peptides, termed ascaphins 1-8, were isolated from norepinephrine-stimulated skin secretions of A. truei and were shown to possess differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus. Ascaphins 2-7 may be represented by the consensus amino acid sequence GX2DX2KGAAKX3KTVAX2IANX.COOH whereas ascaphin-1 (GFRDVLKGAAKAFVKTVAGHIAN.NH2) and ascaphin-8 (GFKDLLKGAAKALVKTVLF.NH2) contain a C-terminally alpha-amidated residue. The ascaphins show no appreciable structural similarity with other families of antimicrobial peptides from frog skin but display limited sequence identity with the cationic, amphipathic alpha-helical peptides pandinin 1 and opistoporin 1, isolated from the venoms of African scorpions. Ascaphin-8 shows the highest potency against a range of pathogenic microorganisms but has the greatest haemolytic activity. The data indicate that the host defence strategy of using antimicrobial peptides in skin secretions arose early in the evolution of anurans.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ranidae/metabolismo , Pele/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/classificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hemólise/efeitos dos fármacos , Humanos , Dose Letal Mediana , Dados de Sequência Molecular , Peso Molecular
10.
Am J Physiol Regul Integr Comp Physiol ; 287(3): R568-74, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15130879

RESUMO

Amylin is postulated to act as a hormonal signal from the pancreas to the brain to inhibit food intake and regulate energy reserves. Amylin potently reduces food intake, body weight, and adiposity when administered systemically or into the brain. Whether selective blockade of endogenous amylin action increases food intake and adiposity remains to be clearly established. In the present study, the amylin receptor antagonist acetyl-[Asn(30), Tyr(32)] sCT-(8-32) (AC187) was used to assess whether action of endogenous amylin is essential for normal satiation to occur. Non-food-deprived rats received a 3- to 4-h intravenous infusion of AC187 (60-2,000 pmol.kg(-1).min(-1)), either alone or coadministered with a 3-h intravenous infusion of amylin (2.5 or 5 pmol.kg(-1).min(-1)) or a 2-h intragastric infusion of an elemental liquid diet (4 kcal/h). Infusions began just before dark onset. Food intake and meal patterns during the first 4 h of the dark period were determined from continuous computer recordings of changes in food bowl weight. Amylin inhibited food intake by approximately 50%, and AC187 attenuated this response by approximately 50%. AC187 dose-dependently stimulated food intake (maximal increases from 76 to 171%), whether administered alone or with an intragastric infusion of liquid diet. Amylin reduced mean meal size and meal frequency, AC187 attenuated these responses, and AC187 administration alone increased mean meal size and meal frequency. These results support the hypothesis that endogenous amylin plays an essential role in reducing meal size and increasing the postmeal interval of satiety.


Assuntos
Amiloide/fisiologia , Ingestão de Alimentos/fisiologia , Receptores de Peptídeos/fisiologia , Amiloide/antagonistas & inibidores , Animais , Anorexia/induzido quimicamente , Anorexia/fisiopatologia , Ingestão de Alimentos/efeitos dos fármacos , Nutrição Enteral , Alimentos Formulados , Injeções Intravenosas , Intubação Gastrointestinal , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Fragmentos de Peptídeos , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas , Receptores de Peptídeos/antagonistas & inibidores
11.
J Med Chem ; 46(12): 2427-35, 2003 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-12773046

RESUMO

Seventeen novel analogues of human calcitonin gene-related peptide(8-37) (hCGRP(8-37)) were synthesized by solid-phase methods and purified to apparent homogeneity by semipreparative cation exchange and/or reversed-phase high-performance liquid chromatography. The C-terminal Phe was replaced by Gly, cyclohexylalanine (Cha), Tyr, all four isomers of beta-methylphenylalanine (beta-MePhe), and l- and d-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the beta-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of beta-MePhe into the erythro pair of enantiomers (2S,3S, 2R,3R) and the threo pair of enantiomers (2S,3R, 2R,3S), which were then converted to Fmoc derivatives and used in two separate syntheses. Two diastereomeric peptides were obtained from each synthesis and were separated by RP-HPLC to yield enantiomerically pure 6-9. Substitution of Tyr for Phe caused no change in binding affinity at CGRP receptors. All other substitutions for Phe resulted in substantial reductions in binding affinity. Indeed, no binding was observed for analogues 7, 9, and 11, all of which contained a d-amino acid residue in the C-terminal position, and the binding affinities of the remaining analogues were >10-fold lower than that of h-alpha-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-alpha-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) produced analogues 12-14 and 16-18, respectively. A byproduct was isolated by RP-HPLC from the resin-cleaved crude product of each benzylated analogue, which was characterized as the dibenzylated derivative of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and (1)H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His(10). Radioligand binding experiments showed that derivatizing the N-termini substantially increased binding affinities at CGRP receptors. The benzoylated and dibenzylated derivatives had the highest affinities, which were approximately 50-fold greater than those of h-alpha-CGRP(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-alpha-CGRP(8-37) are antagonists that are more potent than h-alpha-CGRP(8-37). In conclusion, these studies underscore the importance of Phe(37) of h-alpha-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His(10) as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.


Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/síntese química , Fragmentos de Peptídeos/síntese química , Animais , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Vasos Coronários/metabolismo , Vasos Coronários/fisiologia , Humanos , Técnicas In Vitro , Membranas , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ensaio Radioligante , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/agonistas , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Relação Estrutura-Atividade , Suínos
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