RESUMEN
Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes, or nucleic acids. Polylysine-based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study, we report our findings on the cellular uptake characteristics of nine structurally related polylysine-based polypeptides with cationic side chains composed of (i) single amino acid (poly[Lys(Xi)], XiK) or (ii) oligo[dl-alanine] (poly[Lys(dl-Alam)], AK) or (iii) oligo[dl-alanine] with an additional amino acid (X) at the terminal position (poly[Lys(Xi-dl-Alam)] (XAK)) or (iv) at the position next to the polylysine backbone (poly[Lys(dl-Alam-Xi)] (AXK)). In vitro cytotoxicity and cellular uptake were characterized on HT-29 human colon carcinoma and HepG2 human hepatocarcinoma cell lines. Data indicate that the polycationic polypeptides studied are essentially nontoxic in the concentration range studied, and their uptake is very much dependent on the side chain structure (length, identity of amino acid X, and distance between the terminal positive charges) and also on the cell lines. Our findings in uptake inhibition studies suggest that predominantly macropinocytosis and caveole/lipid raft mediated endocytosis are involved. The efficacy of their internalization is markedly influenced by the hydrophobicity and charge properties of the amino acid X. Interestingly, the uptake properties of the these polypeptides show certain similarities to the entry pathways of several cell penetrating peptides.
Asunto(s)
Péptidos/síntesis química , Péptidos/metabolismo , Polilisina/síntesis química , Polilisina/metabolismo , Cationes , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Endocitosis , Humanos , Péptidos/toxicidad , Polilisina/toxicidad , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Methotrexate (MTX) conjugates with poly[Lys(DL-Alam)] based polymeric polypeptides are efficient against Leishmania donovani parasite infection, but the mechanism of the effect is not known yet. We prepared therefore the 5(6)-carboxyfluorescein (Cf) labeled oligopeptide [Cf-K(AaAa)] (a: D-alanine, A: L-alanine) and the corresponding MTX conjugates [Cf-K(MTX-AaAa)] as model compounds for structure-activity experiments. The conjugate aimed to be synthesized with solid phase methodology on MBHA resin with Boc strategy, using Fmoc-Lys(Boc)-OH. However, various side reactions were identified. Here we report three problems observed during the synthesis as well as solutions developed by us: (1) unexpected cyclopeptide-formation with the lactone-carboxylic group of the Cf was detected, when Cf was attached to the α-amino group of the Lys residue on solid phase. This was avoided by changing the order of Cf incorporation with using Fmoc/Dde strategy. Alternatively, we have built the peptide with Fmoc strategy on solid phase first and performed the labeling with Cf-OSu subsequently in solution. (2) During HF cleavage of the protected conjugates, MTX was demonstrated to form adducts with anisole and p-cresol scavengers, and the TMSOTf cleavage methodology was also found to be inadequate due to the large number of side products formed. We report here that using Fmoc/Dde strategy is an appropriate method to circumvent the cleavage with HF or TMSOTf. (3) During the coupling of MTX with oligopeptide, structural and stereo isomers are formed. We have described here the suitable conditions of HPLC separation of these products.
Asunto(s)
Colorantes Fluorescentes/química , Metotrexato/química , Oligopéptidos , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/aislamiento & purificaciónRESUMEN
Strategies for the preparation of new fluorescent oligopeptide conjugates labeled with 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) at the N-terminal on solid support or in solution have been devised. These procedures are simple and easy to carry out by reacting naOx-OEt or N(alpha)-naOx-amino acid with side chain protected peptide chains attached to resins. The integrity of the N-alkyl bond was maintained even after the trifluoracetic acid or HF based cleavages procedures. Our data show that the naOx fluorophore is compatible with both Fmoc/tBu and Boc/Bzl methods and also suggest that naOx-amino acid could be utilized as building blocks for solid phase peptide synthesis. Comparative analysis of fluorescence properties of naOx-conjugates indicated that the spectral properties of the fluorophore do not change after incorporating into peptides. The compact size, the definite chemical reaction for its introduction in combination with the appropriate spectral features (e.g., intense emission, pH independent fluorescent characteristics, and beneficial photobleaching dose constant and rates) and with chemical and spectral stability, naOx-based labeling could be attractive for novel cellular fluorescent techniques (e.g., in laser scanning confocal FRET) to study peptide-protein and protein-protein interactions even in biological matrices.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Naftalenos/síntesis química , Naftoles/química , Oligopéptidos/síntesis química , Oxazoles/síntesis química , Oxazolona/síntesis química , Alquilación , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Colorantes Fluorescentes/química , Espectrometría de Masas , Datos de Secuencia Molecular , Naftalenos/química , Oligopéptidos/química , Oxazoles/química , Oxazolona/análogos & derivados , Oxazolona/químicaRESUMEN
A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx. 7.0). The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.
Asunto(s)
Colagenasas/aislamiento & purificación , Colagenasas/metabolismo , Photorhabdus/enzimología , Bradiquinina/metabolismo , Cromatografía Líquida de Alta Presión , Colágeno/metabolismo , Colagenasas/química , Cisteína/metabolismo , Gelatina/metabolismo , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis/efectos de los fármacos , Cinética , Espectrometría de Masas , Inhibidores de la Metaloproteinasa de la Matriz , Metales/farmacología , Inhibidores de Proteasas/farmacología , Programas Informáticos , Especificidad por SustratoRESUMEN
his review will summarize available information on the ability of macromolecular conjugates containing no specific recognition motifs to deliver anthracyclines (daunomycin, adriamycin) or methotrexate to target cells such as tumour cells or macrophages. Conjugates with natural (proteins, DNA, carbohydrates) and synthetic macromolecules (linear and branched chain poly-alpha-amino acids, non-biodegradable DIVEMA, HPMA etc.) will be reviewed. Experimental data from several laboratories indicate that these conjugates are taken up by cells mainly by fluid-phase or adsorptive endocytosis. It is believed that these processes do not involve 'specific receptors'. Two examples of methotrexate and daunomycin conjugates will be discussed to show the effect of the chemical structure of branched chain polypeptides on the uptake and antitumour or antiparasitic (Leishmania donovani infection) efficacy of conjugates.
Asunto(s)
Antraciclinas/farmacocinética , Antibióticos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/farmacocinética , Daunorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos , Metotrexato/farmacocinética , Animales , Antraciclinas/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Daunorrubicina/administración & dosificación , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Metotrexato/administración & dosificaciónRESUMEN
Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Ala(m))] (AK), poly[Lys(Ser(i)-DL-Ala(m))] (SAK), poly[Lys(DL-Ala(m)-Leu(i))] (ALK)), or amphoteric (poly[Lys(Glu(i)-DL-Ala(m))] (EAK)) synthetic branched polypeptides containing poly[L-Lys] backbone by the aid of BOP reagent. The average degree of MTX incorporation was found to be dependent on the charge properties of the polymer. Under the experimental conditions used, the molar substitution ratio achieved was higher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect of polycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTX conjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity. In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mice; (b) the covalent bond between the carrier and methotrexate is essential for both in vivo and in vitro activity; and (c) the number of Leishmania donovani parasites in infected macrophages are markedly reduced in conjugate treated animals. In vitro observation might also indicate that the MTX conjugate exhibits an effect through an uptake by macrophages which is different from that of the free drug.