RESUMO
The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.
Assuntos
Anticorpos Monoclonais/metabolismo , Vacinas Anticâncer , Epitopos/imunologia , Espectroscopia de Ressonância Magnética , Análise Serial de Proteínas , Mapeamento de Epitopos , HumanosRESUMO
We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules.
Assuntos
Mitoxantrona/análogos & derivados , RNA/química , Sequência de Bases , Sítios de Ligação , Ligantes , Mitoxantrona/síntese química , Mitoxantrona/química , Estrutura MolecularRESUMO
Tau pre-mRNA contains a stem-loop structure involved in the regulation of the alternative splicing of tau protein. We describe here a new family of Tau RNA ligands selected by dynamic combinatorial chemistry based on the combination of ametantrone with small RNA-binding molecules. The most promising compound results from derivatization of one of the side chains of the anthraquinone ring with the small aminoglycoside neamine through a short spacer. This compound binds the RNA target with a high affinity in a preferred binding site, in which the heteroaromatic moiety intercalates in the bulged region of the stem-loop and its side chains and neamine interact with the major groove of the RNA. Importantly, binding of this compound to mutated RNA sequences involved in the onset of some tauopathies such as FTDP-17 restores their thermodynamic stability to a similar or even higher levels than that of the wild-type sequence, thereby revealing its potential as a modulator of Tau pre-mRNA splicing.
Assuntos
Processamento Alternativo , Mitoxantrona/análogos & derivados , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas tau/genética , Espectroscopia de Ressonância Magnética , Mitoxantrona/químicaRESUMO
A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η(6)-p-cym)RuCl(PPh3)2](+), allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-Nâ³-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 µM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 µM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 µM in DU-145 and IC50 = 11.33 µM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 â« 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 µM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.
Assuntos
Antineoplásicos/química , Neomicina/análogos & derivados , Compostos Organometálicos/química , Rutênio/química , Aminoglicosídeos/química , Aminoglicosídeos/farmacocinética , Aminoglicosídeos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Transporte Biológico Ativo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Células MCF-7 , Masculino , Neomicina/farmacocinética , Neomicina/farmacologia , Compostos Organometálicos/farmacocinética , Compostos Organometálicos/farmacologia , RNA Neoplásico/efeitos dos fármacos , RNA Neoplásico/metabolismo , Rutênio/farmacocinética , Rutênio/farmacologiaRESUMO
The design and synthesis of two Janus-type heterocycles with the capacity to simultaneously recognize guanine and uracyl in G-U mismatched pairs through complementary hydrogen bond pairing is described. Both compounds were conveniently functionalized with a carboxylic function and efficiently attached to a tripeptide sequence by using solid-phase methodologies. Ligands based on the derivatization of such Janus compounds with a small aminoglycoside, neamine, and its guanidinylated analogue have been synthesized, and their interaction with Tau RNA has been investigated by using several biophysical techniques, including UV-monitored melting curves, fluorescence titration experiments, and (1)H NMR. The overall results indicated that Janus-neamine/guanidinoneamine showed some preference for the +3 mutated RNA sequence associated with the development of some tauopathies, although preliminary NMR studies have not confirmed binding to G-U pairs. Moreover, a good correlation has been found between the RNA binding affinity of such Janus-containing ligands and their ability to stabilize this secondary structure upon complexation.
Assuntos
Guanina/química , RNA/química , Uracila/química , Pareamento Incorreto de Bases , Pareamento de Bases , Dicroísmo Circular , Ligação de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
We describe the effect of guanidinylation of the aminoglycoside moiety on acridine-neamine-containing ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, an important regulatory element of tau gene alternative splicing. On the basis of dynamic combinatorial chemistry experiments, ligands that combine guanidinoneamine and two different acridines were synthesized and their RNA-binding properties were compared with those of their amino precursors. Fluorescence titration experiments and UV-monitored melting curves revealed that guanidinylation has a positive effect both on the binding affinity and specificity of the ligands for the stem-loop RNA, as well as on the stabilization of all RNA sequences evaluated, particularly some mutated sequences associated with the development of FTDP-17 tauopathy. However, this correlation between binding affinity and stabilization due to guanidinylation was only found in ligands containing a longer spacer between the acridine and guanidinoneamine moieties, since a shorter spacer produced the opposite effect (e.g. lower binding affinity and lower stabilization). Furthermore, spectroscopic studies suggest that ligand binding does not significantly change the overall RNA structure upon binding (circular dichroism) and that the acridine moiety might intercalate near the bulged region of the stem-loop structure (UV-Vis and NMR spectroscopy).
Assuntos
Acridinas/síntese química , Aminoglicosídeos/química , Guanidinas/síntese química , Precursores de RNA/química , Proteínas tau/química , Processamento Alternativo , Sequência de Bases , Dicroísmo Circular , Técnicas de Química Combinatória , Éxons , Humanos , Íntrons , Sequências Repetidas Invertidas , Ligantes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estabilidade de RNA , Sequências Reguladoras de Ácido Nucleico , Tauopatias/genéticaRESUMO
In many human carcinomas, mucin-1 (MUC1) is overexpressed and aberrantly glycosylated, resulting in the exposure of previously hidden antigens. This generates new patient antibody profiles that can be used in cancer diagnosis. In the present study, we focused on the MUC1-associated Tn antigen (α-O-GalNAc-Ser/Thr) and substituted the GalNAc monosaccharide by a glycomimic to identify MUC1-based glycopeptides with increased antigenicity. Two different glycopeptide libraries presenting the natural Tn antigen or the sp2-iminosugar analogue were synthesized and evaluated with anti-MUC1 monoclonal antibodies in a microarray platform. The most promising candidates were tested with healthy and breast cancer sera aiming for potential autoantibody-based biomarkers. The suitability of sp2-iminosugar glycopeptides to detect anti-MUC1 antibodies was demonstrated, and serological experiments showed stage I breast cancer autoantibodies binding with a specific unnatural glycopeptide with almost no healthy serum interaction. These results will promote further studies on their capabilities as early cancer biomarkers.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Autoanticorpos/imunologia , Neoplasias da Mama/imunologia , Mucina-1/imunologia , Antígenos Glicosídicos Associados a Tumores/química , Autoanticorpos/sangue , Neoplasias da Mama/sangue , Feminino , Glicômica , Humanos , Mucina-1/química , Biblioteca de PeptídeosRESUMO
Functional pairing of cellular glycoconjugates with tissue lectins is a highly selective process, whose determinative factors have not yet been fully delineated. Glycan structure and modes of presentation, that is, its position and density, can contribute to binding, as different members of a lectin family can regulate degrees of responsiveness to these factors. Using a peptide repeat sequence motif of the glycoprotein mucin-1, the principle of introducing synthetic (glyco)peptides with distinct variations in these three parameters to an array-based screening of tissue lectins is illustrated. Interaction profiles of seven adhesion/growth-regulatory galectins cover the range from intense signals with core 2 pentasaccharides and core 1 binding for galectins-3 and -5 to a lack of binding for galectin-1 and also the galectin-related protein, which was included as a negative control. Remarkably, the two tandem-repeat-type galectins-4 and -8 were distinguished by core 1 sialylation, as the two separated domains were. These results encourage further synthetic elaboration of the glycopeptide library and testing of the network of natural galectins and rationally engineered variants of the lectins.
Assuntos
Galectinas/química , Glicopeptídeos/síntese química , Polissacarídeos/química , Galectinas/metabolismo , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Conformação Molecular , Biblioteca de Peptídeos , Polissacarídeos/metabolismoRESUMO
The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL.
Assuntos
Células Dendríticas/imunologia , Glicopeptídeos/imunologia , Lectinas Tipo C/metabolismo , Mucina-1/metabolismo , Animais , Glicosilação , Humanos , Lectinas Tipo C/química , Camundongos , Ligação ProteicaRESUMO
Conjugates of a Pt(IV) derivative of picoplatin with monomeric (Pt-c(RGDfK), 5) and tetrameric (Pt-RAFT-{c(RGDfK)}4, 6) RGD-containing peptides were synthesized with the aim of exploiting their selectivity and high affinity for αVß3 and αVß5 integrins for targeted delivery of this anticancer metallodrug to tumor cells overexpressing these receptors. Solid- and solution-phase approaches in combination with click chemistry were used for the preparation of the conjugates, which were characterized by high resolution ESI MS and NMR. αVß3 and αVß5 integrin expression was evaluated in a broad panel of human cancer and non-malignant cells. SK-MEL-28 melanoma cells were selected based on the high expression levels of both integrins, while CAPAN-1 pancreatic cancer cells and 1BR3G fibroblasts were selected as the negative control. Internalization experiments revealed a good correlation between integrin expression and the cellular uptake of the corresponding fluorescein-labeled peptides and that the internalization capacity of the tetrameric RGD-containing peptide was considerably higher than that of the monomeric one. Cytotoxic experiments indicated that the antitumor activity of picoplatin in melanoma cells was increased by 2.6-fold when its Pt(IV) derivative was conjugated to c(RGDfK) (IC50 = 12.8 ± 2.1 µM) and by 20-fold when conjugated to RAFT-{c(RGDfK)}4 (IC50 = 1.7 ± 0.6 µM). In contrast, the cytotoxicity of the conjugates was inhibited in control cells lacking αVß3 and αVß5 integrin expression. Finally, cellular uptake studies by ICP-MS confirmed a good correlation between the levels of expression of integrins, intracellular platinum accumulation and antitumor activity. Indeed, accumulation and cytotoxicity were much higher in SK-MEL-28 cells than in CAPAN-1, being particularly higher in the case of the tetrameric conjugate. The overall results highlight that the great ability of RAFT-{c(RGDfK)}4 to bind to and to be internalized by integrins overexpressed in SK-MEL-28 cells results in higher accumulation of the Pt(IV) complex, leading to a high antitumor activity. These studies provide new insights into the potential of targeting αVß3 and αVß5 integrins with Pt(IV) anticancer pro-drugs conjugated to tumor-targeting devices based on RGD-containing peptides, particularly on how multivalency can improve both the selectivity and potency of such metallodrugs by increasing cellular accumulation in tumor tissues.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Integrina alfaVbeta3/metabolismo , Oligopeptídeos/administração & dosagem , Compostos Organoplatínicos/administração & dosagem , Pró-Fármacos/administração & dosagem , Receptores de Vitronectina/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacocinética , Compostos Organoplatínicos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologiaRESUMO
A new anticancer agent based on the conjugation of a photoactivatable Pt(IV) pro-drug to a cyclic RGD-containing peptide is described. Upon visible light irradiation, phototoxicity was induced preferentially in SK-MEL-28 melanoma cancer cells overexpressing αVß3 integrin compared to control DU-145 human prostate carcinoma cells.