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1.
J Med Chem ; 63(23): 14668-14679, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33226806

RESUMO

Minigastrin (MG) analogues, known for their high potential to target cholecystokinin-2 receptor (CCK2R) expressing tumors, have limited clinical applicability due to low enzymatic stability. By introducing site-specific substitutions within the C-terminal receptor-binding sequence, reduced metabolization and improved tumor targeting can be achieved. In this work, the influence of additional modification within the N-terminal sequence has been explored. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R ligands with proline substitution at different positions were synthesized. Substitution did not affect CCK2R affinity, and the conjugates labeled with indium-111 and lutetium-177 showed a high enzymatic stability in different incubation media as well as in vivo (57-79% intact radiopeptide in blood of BALB/c mice at 1 h p.i.) combined with enhanced tumor uptake (29-46% IA/g at 4 h in xenografted BALB/c nude mice). The inclusion of Pro contributes significantly to the development of CCK2R ligands with optimal targeting properties for application in targeted radiotherapy.


Assuntos
Gastrinas/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Prolina/química , Compostos Radiofarmacêuticos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Feminino , Gastrinas/síntese química , Gastrinas/farmacocinética , Compostos Heterocíclicos com 1 Anel/síntese química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Radioisótopos de Índio/química , Lutécio/química , Camundongos Endogâmicos BALB C , Ligação Proteica , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Receptor de Colecistocinina B/metabolismo
2.
J Med Chem ; 63(9): 4496-4505, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302130

RESUMO

The insertion of single 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres of trans-amide bonds (triazole scan) was recently applied to the 177Lu-labeled tumor-targeting analog of minigastrin, [Nle15]MG11. The reported novel mono-triazolo-peptidomimetics of [Nle15]MG11 showed either improved resistance against enzymatic degradation or a significantly increased affinity toward the target receptor but never both. To enhance further the tumor-targeting properties of the minigastrin analogs, we studied conjugates with multiple amide-to-triazole substitutions for additive or synergistic effects. Promising candidates were identified by modification of two or three amide bonds, which yielded both improved stability and increased receptor affinity of the peptidomimetics in vitro. Biodistribution studies of radiolabeled multi-triazolo-peptidomimetics in mice bearing receptor-positive tumor xenografts revealed up to 4-fold increased tumor uptake in comparison to the all-amide reference compound [Nle15]MG11. In addition, we report here for the first time a linear peptidomimetic with three triazole insertions in its backbone and maintained biological activity.


Assuntos
Antineoplásicos/farmacologia , Gastrinas/farmacologia , Peptidomiméticos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Gastrinas/síntese química , Gastrinas/metabolismo , Gastrinas/farmacocinética , Humanos , Lutécio/química , Camundongos , Neoplasias/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Peptidomiméticos/farmacocinética , Ligação Proteica , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Colecistocinina B/metabolismo , Triazóis/síntese química , Triazóis/metabolismo , Triazóis/farmacocinética
3.
J Med Chem ; 63(9): 4484-4495, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302139

RESUMO

MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer diagnosis and therapy. A drawback of MG11 is its fast degradation by proteases, leading to moderate tumor uptake in vivo. We introduced 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres to replace labile amide bonds of the peptide. The "triazole scan" yielded peptidomimetics with improved resistance to enzymatic degradation and/or enhanced affinity toward the CCK2R. Remarkably, our lead compound achieved a 10-fold increase in receptor affinity, resulting in a 2.6-fold improved tumor uptake in vivo. Modeling of the ligand-CCK2R complex suggests that an additional cation-π interaction of the aromatic triazole moiety with the Arg356 residue of the receptor is accountable for these observations. We show for the first time that the amide-to-triazole substitution strategy offers new opportunities in drug development that go beyond the metabolic stabilization of bioactive peptides.


Assuntos
Antineoplásicos/farmacologia , Gastrinas/farmacologia , Peptidomiméticos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Feminino , Gastrinas/síntese química , Gastrinas/metabolismo , Gastrinas/farmacocinética , Humanos , Lutécio/química , Camundongos , Neoplasias/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Peptidomiméticos/farmacocinética , Ligação Proteica , Conformação Proteica , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Colecistocinina B/metabolismo , Triazóis/síntese química , Triazóis/metabolismo , Triazóis/farmacocinética
4.
J Pept Sci ; 23(1): 38-44, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28054429

RESUMO

Radiolabelled peptides with high specificity and affinity towards receptors that are overexpressed by tumour cells are used in nuclear medicine for the diagnosis (imaging) and therapy of cancer. In some cases, the sequences of peptides under investigations contain methionine (Met), an amino acid prone to oxidation during radiolabelling procedures. The formation of oxidative side products can affect the purity of the final radiopharmaceutical product and/or impair its specificity and affinity towards the corresponding receptor. The replacement of Met with oxidation resistant amino acid analogues, for example, norleucine (Nle), can provide a solution. While this approach has been applied successfully to different radiolabelled peptides, a Met → Nle switch only preserves the length of the amino acid side chain important for hydrophobic interactions but not its hydrogen-bonding properties. We report here the use of methoxinine (Mox), a non-canonical amino acid that resembles more closely the electronic properties of Met in comparison to Nle. Specifically, we replaced Met15 by Mox15 and Nle15 in the binding sequence of a radiometal-labelled human gastrin derivative [d-Glu10 ]HG(10-17), named MG11 (d-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ). A comparison of the physicochemical properties of 177 Lu-DOTA[X15 ]MG11 (X = Met, Nle, Mox) in vitro (cell internalization/externalization properties, receptor affinity (IC50 ), blood plasma stability and logD) showed that Mox indeed represents a suitable, oxidation-stable amino acid substitute of Met in radiolabelled peptide conjugates. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Gastrinas/síntese química , Compostos Heterocíclicos com 1 Anel/química , Homosserina/análogos & derivados , Lutécio/química , Oligopeptídeos/síntese química , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Substituição de Aminoácidos , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Gastrinas/metabolismo , Gastrinas/farmacologia , Compostos Heterocíclicos com 1 Anel/metabolismo , Compostos Heterocíclicos com 1 Anel/farmacologia , Homosserina/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Metionina/química , Norleucina/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Oxirredução , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacologia , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo
5.
Peptides ; 74: 16-22, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26471904

RESUMO

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function.


Assuntos
Gastrinas/química , Gastrinas/farmacologia , Fármacos Gastrointestinais/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Gastrinas/síntese química , Fármacos Gastrointestinais/síntese química , Fármacos Gastrointestinais/farmacologia , Humanos , Íons/química , Ferro/química , Camundongos , Fragmentos de Peptídeos/síntese química
6.
Pak J Pharm Sci ; 26(2): 299-305, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23455200

RESUMO

Radiolabeled neuropeptides are widely investigated to diagnose and therapy of tumors. These peptides get internalization after binding with particular receptors at the surface of cells and finally move to lysosome. Internalization into tumor cells helps in mapping the infected site. Minigastrin peptide analogues (MG-CL1-4) were synthesised and labeled with 111-In radioisotope under different sets of conditions for imaging CCk-2 receptor bearing tumors. Different parameters such as temperature (80-100°C), pH (4-12), incubation time (5-30 minutes) and dilution effect were investigated to get the maximum labeling yield and stability. The results indicated that MG-CL1-4 is successfully labeled with indium-111 at pH 4.5 with heating at 98°C for 15 minute. At these conditions i.e. heating, pH and incubation minimum oxidized and maximum labeling yield, more than 94 %, was obtained. The labeling stability was studied by incubating the radiolabeled complex for predefined time points in PBSA and blood serum. Results show that more than 90% radiolabeled MG-CL1-4 remained intact.


Assuntos
Gastrinas/síntese química , Radioisótopos de Índio , Marcação por Isótopo , Gastrinas/sangue , Gastrinas/normas , Humanos , Concentração de Íons de Hidrogênio , Marcação por Isótopo/normas , Oxirredução , Estabilidade Proteica , Controle de Qualidade , Temperatura , Fatores de Tempo
7.
J Med Chem ; 52(15): 4786-93, 2009 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-19591486

RESUMO

Two cyclized minigastrin analogues for gastrin receptor scintigraphy were synthesized and derivatized with HYNIC at the N-terminus for labeling with 99mTc. Radiolabeling efficiency, stability, cell internalization, and receptor binding on CCK-2 receptor expressing AR42J cells were studied and the biodistribution evaluated in tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver, and kidneys were analyzed by radio-HPLC. Radiolabeled cyclic MG showed high stability in vitro and receptor mediated uptake in AR42J cells. In the animal tumor model, fast renal clearance and low nonspecific uptake in most organs were observed. A tumor uptake >3% was calculated ex vivo 1 h p.i. for both 99mTc-EDDA-HYNIC-cyclo-MG1 and 99mTc-EDDA-HYNIC-cyclo-MG2. In an imaging study with 99mTc-EDDA-HYNIC-cyclo-MG1, the tumor was clearly visualized. The metabolite analysis indicated rapid enzymatic degradation in vivo.


Assuntos
Gastrinas/síntese química , Compostos Radiofarmacêuticos/síntese química , Receptor de Colecistocinina B/análise , Tecnécio , Sequência de Aminoácidos , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Gastrinas/farmacocinética , Marcação por Isótopo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neoplasias Experimentais/metabolismo , Distribuição Tecidual
8.
Eur J Pharm Sci ; 31(2): 102-11, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17387005

RESUMO

A DOTA-gastrin analogue (APH070) which, when labelled with (111)In, has high affinity for the gastrin/CCK-2 receptor (3nM) and low tumour to kidney ratio in animal models, has been formulated and manufactured for a clinical study. Oxidation of the peptide methionine residue greatly reduces receptor affinity, therefore development work focused on producing a stable intermediate drug product (iDP) whilst ensuring that the formulation, container, closure and manufacturing process did not inhibit the extremely sensitive radiolabelling reaction (itself a source of oxidation). Stress testing revealed that APH070 was stable at 2-8 degrees C at pH 6-9. Addition of an antioxidant (monothioglycerol) to the peptide formulation reduced stability when compared to buffer alone. Use of FluroTec (4023/50) stoppers (rather than FluroTec Plus (4110/40)) increased both the stability and radiolabelling efficiency of APH070. Long term stability (6 months) of the final formulation (1mg/ml APH070 in 0.01 M pH 7.2 phosphate buffer) stored at 5 degrees C in type I glass vials with FluroTec (4023/50) stoppers was 98.6+/-0.2% and 98.4+/-0.1% for upright and inverted samples, respectively. Clinical scale radiolabelling of the final formulation routinely achieves the specification of >85% (111)In-APH070 (unoxidised) stable for up to 2h after dilution with 0.9% w/v saline solution. Specific uptake of the radiopharmaceutical in CCK-2R-expressing AR42J tumours in nude mice has been demonstrated.


Assuntos
Embalagem de Medicamentos , Gastrinas/síntese química , Radioisótopos de Índio , Marcação por Isótopo , Oligopeptídeos/síntese química , Compostos Radiofarmacêuticos/síntese química , Receptor de Colecistocinina B/metabolismo , Tecnologia Farmacêutica/métodos , Animais , Linhagem Celular Tumoral , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Gastrinas/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Metionina/química , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Oligopeptídeos/metabolismo , Oxirredução , Compostos Radiofarmacêuticos/metabolismo , Reprodutibilidade dos Testes , Solubilidade , Soluções , Temperatura , Fatores de Tempo
9.
J Med Chem ; 50(6): 1418-22, 2007 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-17315986

RESUMO

Fmoc-lys(HYNIC-Boc)-OH, a precursor for solid-phase synthesis of 99mTc-labeled peptides, was synthesized efficiently without HPLC purification. HPLC-ESMS showed that deprotection and decoupling of peptide from the resin with trifluoroacetic acid gave initially HYNIC-peptide, which was trifluoroacetylated upon prolonged incubation. The trifluoroacetyl-HYNIC group was hydrolyzed during 99mTc labeling, rendering deprotection unnecessary. Trifluoroacetyl-HYNIC peptide was 99mTc-labeled as efficiently, producing the same product, as HYNIC-peptide. These modifications enhance the versatility of HYNIC for 99mTc peptide labeling.


Assuntos
Fluoracetatos , Gastrinas/síntese química , Hidrazinas/síntese química , Ácidos Nicotínicos/síntese química , Oligopeptídeos/síntese química , Fragmentos de Peptídeos/síntese química , Compostos Radiofarmacêuticos/síntese química , Tecnécio , Ácido Trifluoracético/síntese química , Gastrinas/química , Hidrazinas/química , Hidrólise , Marcação por Isótopo , Ácidos Nicotínicos/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Compostos Radiofarmacêuticos/química , Ácido Trifluoracético/química
10.
Chem Pharm Bull (Tokyo) ; 49(8): 958-63, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11515585

RESUMO

Application of the fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase segment condensation approach to the preparation of sulfated peptides was investigated through the synthesis of human big gastrin-II, a 34-residue sulfated tyrosine [Tyr(SO3H)]-containing peptide. Highly acid-sensitive 2-chlorotrityl resin (Clt resin) was exclusively employed as an anchor-resin for the preparation of the three peptide segments having the C-terminal Pro residue as well as of the Tyr(SO3H)-containing resin-bound segment. By using the PyBOP-mediated coupling protocol [PyBOP=benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphatel, we successively condensed each segment and constructed the 34-residue peptide-resin without any difficulty. The final acid treatment of the fully protected peptide-resin at low temperature (90% aqueous TFA, 0 degree C for 8 h), which can detach a Tyr(SO3H)-containing peptide from the resin and remove the protecting groups concurrently with minimum deterioration of the sulfate, afforded a crude sulfated peptide. After one-step HPLC purification, a highly homogeneous human big gastrin-II was easily obtained in 14% yield from the protected peptide-resin. The sulfate form of the C-terminal glycine-extended gastrin (G34-Gly sulfate), a posttranslational processing intermediate of gastrin-II, was also successfully prepared with the segment condensation approach (11% yield). These results demonstrated the usefulness of the segment condensation protocol for preparing large Tyr(SO3H)-containing peptides.


Assuntos
Gastrinas/síntese química , Glicina/síntese química , Peptídeos/síntese química , Precursores de Proteínas/síntese química , Sulfatos/síntese química , Tirosina/síntese química , Humanos , Processamento de Proteína Pós-Traducional
11.
J Org Chem ; 66(1): 1-10, 2001 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-11429884

RESUMO

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.


Assuntos
Colecistocinina/análogos & derivados , Colecistocinina/síntese química , Gastrinas/síntese química , Peptídeos/síntese química , Precursores de Proteínas/síntese química , Tirosina/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Hidrólise , Técnicas In Vitro , Indicadores e Reagentes , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , Peptídeos/química , Ratos , Serina Endopeptidases/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sulfatos/química , Água/química
12.
Proc Natl Acad Sci U S A ; 95(20): 11520-5, 1998 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-9751698

RESUMO

In principle, cell surface receptors that are overexpressed in tumor tissue could serve as targets for anticancer drugs attached to receptor ligands. The purpose of this paper is to identify the necessary elements for a successful receptor-targeted drug. We used the gastrin/cholecystokinin type B receptor as a model delivery system, and we report on the synthesis, trafficking, and in vitro and in vivo evaluation of heptagastrin, the C-terminal heptapeptide of gastrin, linked via an appropriate linker to a potently cytotoxic ellipticine derivative, 1-[3-[N-(3-aminopropyl)-N-methylamino]propyl]amino-9-methoxy-5, 11-dimethyl-6H-pyrido[4,3-b]carbazole. These data, and previous work from our laboratory, show that the drug-complexed ligand is sorted to lysosomes whereas the receptor is recycled to the plasma membrane. The lysosomal processing of the ligand/drug construct depends on the linker between the ligand sequence and the cytotoxic moiety. We show that heptagastrin linked to ellipticine via a succinoyl-substituted pentapeptide, AlaLeuAlaLeuAla, is at least 10(3) more toxic to cholecystokinin type B receptor-positive NIH/3T3 cells than to isogenic NIH/3T3 cells lacking the receptor. The conjugated drug eradicated all receptor-positive tumor cells in vivo without producing any general toxicity. The data indicate that the density of the cell surface receptor, the properties of the cytotoxic moiety, and the correct processing of the drug-conjugated ligand in lysosomes are crucial to the effectiveness of a receptor-targeted drug.


Assuntos
Antineoplásicos/farmacologia , Receptores da Colecistocinina/efeitos dos fármacos , Células 3T3 , Sequência de Aminoácidos , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Cálcio/metabolismo , Membrana Celular/metabolismo , Elipticinas/síntese química , Elipticinas/química , Elipticinas/farmacologia , Gastrinas/síntese química , Gastrinas/química , Gastrinas/farmacologia , Humanos , Cinética , Ligantes , Lisossomos/metabolismo , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Receptor de Colecistocinina B , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/metabolismo , Transfecção
13.
Int J Pept Protein Res ; 44(4): 348-56, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7875937

RESUMO

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, 125I-BH-[Leu15]-gastrin-(5-17) [(3-[125I]iodo-4-hydroxyphenyl)-propionyl-[Leu15]-gastrin-(5-17)]. Binding of 125I-BH-[Leu15]-gastrin-(5-17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. 125I-BH-[Leu15]-gastrin- (5-17) and 125I-BH-CCK-8[(3-[125I]iodo-4-hydroxyphenyl)-propionyl-CCK-8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 +/- 900 binding sites/cell (Kd = 0.52 +/- 0.23 nM) and 6200 +/- 1100 binding sites/cell (Kd = 0.31 +/- 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing 125I-BH-[Leu15]-gastrin-(5-17) binding correlated well with those obtained using 125I-BH-CCK-8. Selective CCK/gastrin antagonists L-364,718 (MK-329) and L-365,260 also inhibited 125I-BH-[Leu15]-gastrin-(5-17) binding. These results indicate that 125I-BH-[Leu15]-gastrin-(5-17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized 125I-BH-[Leu15]-gastrin-(5-17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK-B/gastrin receptor. Saturation experiments have shown that both 125I-BH-[Leu15]-gastrin-(5-17) and 125I-BH-CCK-8 interact with a single class of high-affinity binding sites in the Jurkat cell line.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Mucosa Gástrica/metabolismo , Gastrinas/síntese química , Gastrinas/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Compostos de Fenilureia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Benzodiazepinonas/farmacologia , Cálcio/metabolismo , Linhagem Celular , Colecistocinina/antagonistas & inibidores , Devazepida , Cães , Fundo Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Gastrinas/farmacologia , Humanos , Radioisótopos do Iodo , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Sincalida/farmacologia , Linfócitos T/efeitos dos fármacos , Temperatura
14.
Int J Pept Protein Res ; 38(6): 555-61, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1819590

RESUMO

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.


Assuntos
Gastrinas/síntese química , Sincalida/síntese química , Sequência de Aminoácidos , Aminoácidos , Fluorenos , Gastrinas/química , Dados de Sequência Molecular , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Resinas Sintéticas , Sincalida/química , Tirosina/química
15.
J Biol Chem ; 266(35): 24126-33, 1991 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1721065

RESUMO

Treatment of quiescent Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, endothelin/vasoactive intestinal contractor (VIC), and bradykinin strikingly increased the initial rate of tyrosine phosphorylation measured in anti-phosphotyrosine immunoprecipitates of a major band of Mr 115,000 (p115) and two minor components of Mr 90,000 and 75,000. Neuropeptides increased the labeling of p115 within seconds and with great potency; half-maximum concentrations were 0.1, 0.2 and 0.3 nM for bombesin, vasopressin, and VIC, respectively. Immunoblotting and peptide mapping showed that the p115 band phosphorylated in anti-phosphotyrosine immunoprecipitates is identical to a major Mr 115,000 substrate for neuropeptide-stimulated tyrosine phosphorylation in intact Swiss 3T3 cells. Furthermore, bombesin, vasopressin, and VIC markedly increased the rate of phosphorylation of Raytide, a broad specificity tyrosine kinase peptide substrate, by decreasing (8 +/- 1.3-fold) the apparent Km of the kinase for the substrate. Phorbol 12,13-dibutyrate and the Ca2+ ionophore A23187 had a weaker effect on tyrosine protein kinase activity in immune complexes compared with bombesin. Furthermore, down-regulation of protein kinase C blocked the small effect of phorbol esters but did not impair bombesin-stimulated tyrosine kinase activity. These results provide direct evidence for neuropeptide activation of a tyrosine kinase in cell-free preparations and identify a novel event in the action of this class of growth factors in Swiss 3T3 cells.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Bombesina/farmacologia , Endotelinas/farmacologia , Neuropeptídeos/farmacologia , Proteínas Tirosina Quinases/metabolismo , Tirosina/análogos & derivados , Vasopressinas/farmacologia , Células 3T3 , Sequência de Aminoácidos , Animais , Calcimicina/farmacologia , Sistema Livre de Células , Gastrinas/síntese química , Gastrinas/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Fosfotirosina , Especificidade por Substrato , Tirosina/imunologia , Vanadatos/farmacologia
16.
Biol Chem Hoppe Seyler ; 372(3): 163-72, 1991 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2054095

RESUMO

Two N-terminal biotinyl-gastrin derivatives were synthesized to investigate the effect of the length and chemical properties of the biotin-spacer on both the capture of the hapten by streptavidin or avidin adsorbed on polystyrene, and the antigenicity of the captured peptide. The observed full retainment of antibody binding capacity of the biotinyl-gastrins upon their immobilization, allowed to develop a sandwich-type ELISA with a sensitivity of one order of magnitude better than the standard ELISA with polystyrene-adsorbed gastrin. This hapten capture system reduces desorption particularly pronounced for low mass peptides, and avoids possible modifications or suppression of epitopes by the adsorption process with concomitant reduction of antibody binding affinity of the antigen. This new type of assay procedure may also represent a useful tool particularly for epitope mapping with relatively low mass synthetic protein fragments.


Assuntos
Gastrinas/análise , Haptenos , Sequência de Aminoácidos , Avidina/análise , Proteínas de Bactérias/análise , Biotina/análise , Biotina/síntese química , Ensaio de Imunoadsorção Enzimática , Gastrinas/síntese química , Haptenos/síntese química , Humanos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Estreptavidina
17.
Int J Pept Protein Res ; 37(2): 90-102, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1708369

RESUMO

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.


Assuntos
Gastrinas/síntese química , Gastrinas/imunologia , Imunoglobulina G/síntese química , Fragmentos de Peptídeos/síntese química , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Gastrinas/química , Cobaias , Haptenos/síntese química , Haptenos/imunologia , Humanos , Imunização , Imunoglobulina G/química , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Conformação Proteica
18.
Int J Pept Protein Res ; 37(1): 61-71, 1991 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2045221

RESUMO

The bis-cysteinyl hinge-fragment 225-232 of human IgG1 has been extended at the N- or C-terminus with Nle15-desamido-human-little-gastrin-[5-17] and Nle15-human-little-gastrin-[5-17]-NH2, respectively. Thermodynamically controlled air oxidation of the resulting bis-cysteinyl-peptides led to the predominant formation of the corresponding dimers in parallel alignment despite the incorporation of the immunoglobulin-unrelated gastrin-sequences. These surprising results confirm the high degree of structural information inherent in the hinge-sequence and its intrinsic tendency to fold into the correct structure in terms of cysteine pairings. This protein subdomain-the hinge-peptide-is therefore well suited as core molecule for the design of fully synthetic immunogens with multiple attachment of antigenic determinants.


Assuntos
Antígenos/síntese química , Peptídeos/síntese química , Sequência de Aminoácidos , Antígenos/química , Gastrinas/síntese química , Gastrinas/química , Gastrinas/imunologia , Humanos , Imunoglobulina G/síntese química , Imunoglobulina G/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica
19.
J Biol Chem ; 265(27): 16205-9, 1990 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-2168884

RESUMO

Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation.


Assuntos
Gastrinas/síntese química , Oligopeptídeos/síntese química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Desenho de Fármacos , Gastrinas/metabolismo , Gastrinas/farmacologia , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Fosforilação , Receptor de Insulina
20.
Int J Pept Protein Res ; 35(6): 527-38, 1990 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2401594

RESUMO

An approach to the solid-phase segment condensation synthesis of the 17-peptide amide human gastrin-I has been developed. N alpha-amino and side-chain protection were provided by 9-fluorenylmethyloxycarbonyl (Fmoc) and tert.-butyl groups, and a series of anchors cleavable under mild conditions were used. The N-terminal pentapeptide pGlu-Gly-Pro-Trp-Leu-OH was prepared using a p-alkoxybenzyl ester linkage made by a preformed handle strategy. Cleavage, in 65% yield, was with the new Reagent M: CF3COOH-CH2Cl2-beta-mercaptoethanol--anisole (70:30:2:1), which was optimized to preserve the labile tryptophan residue. A new preformed handle procedure expedited solid-phase synthesis of the protected "middle" hexapeptide, Fmoc-(Glu(OtBu]5-Ala-OH, anchored as an o-nitrobenzyl ester. Chains were not lost during this assembly, and final photolytic cleavage (350 nm) in toluene--CF3CH2OH (4:1) occurred in 59% yield. Both protected intermediates were purified by simple gel filtration, whereupon they were shown to be pure by analytical HPLC, and gave satisfactory NMR and FABMS spectra. Last, the C-terminal hexapeptide, Tyr(tBu)-Gly-Trp-Met-Asp(OtBu)-Phe, was assembled on a tris(alkoxy)benzylamide "PAL" support. For the polymer-supported segment condensation, the middle and N-terminal pieces were added respectively in greater than 98% and 89% yields (judged by amino acid analysis and solid-phase sequencing), by overnight couplings in N,N-dimethylformamide (DMF) mediated by benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). Racemization was 4% and 11% respectively at Ala and Leu. Cleavage with Reagent M followed by reversed-phase chromatography gave pure gastrin-I in an overall 30% isolated yield. These results compare favorably with those from a stepwise assembly.


Assuntos
Gastrinas/síntese química , Sequência de Aminoácidos , Fenômenos Químicos , Química , Humanos , Indicadores e Reagentes , Dados de Sequência Molecular
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