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Aminopeptidase N/CD13, a membrane-bound enzyme upregulated in tumor vasculature and involved in angiogenesis, can be used as a receptor for the targeted delivery of drugs to tumors through ligand-directed targeting approaches. We describe a novel peptide ligand (VGCARRYCS, called "G4") that recognizes CD13 with high affinity and selectivity. Enzymological and computational studies showed that G4 is a competitive inhibitor that binds to the catalytic pocket of CD13 through its N-terminal region. Fusing the peptide C-terminus to tumor necrosis factor-alpha (TNF) or coupling it to a biotin/avidin complex causes loss of binding and inhibitory activity against different forms of CD13, including natural or recombinant ectoenzyme and a membrane form expressed by HL60 promyelocytic leukemia cells (likely due to steric hindrance), but not binding to a membrane form of CD13 expressed by endothelial cells (ECs). Furthermore, G4-TNF systemically administered to tumor-bearing mice exerted anticancer effects through a CD13-targeting mechanism, indicating the presence of a CD13 form in tumor vessels with an accessible binding site. Biochemical studies showed that most CD13 molecules expressed on the surface of ECs are catalytically inactive. Other functional assays showed that these molecules can promote endothelial cell adhesion to plates coated with G4-avidin complexes, suggesting that the endothelial form of CD13 can exert catalytically independent biological functions. In conclusion, ECs express a catalytically inactive form of CD13 characterized by an accessible conformation that can be selectively targeted by G4-protein conjugates. This form of CD13 may represent a specific target receptor for ligand-directed targeted delivery of therapeutics to tumors.
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Antígenos CD13 , Células Endoteliais , Leucemia Promielocítica Aguda , Animais , Camundongos , Antígenos CD13/antagonistas & inibidores , LigantesRESUMO
Supramolecular hydrogels play a pivotal role in many fields of biomedical research, including emerging applications in designing advanced tools for point-of-care testing, clinical diagnostics, and lab-on-chip analysis. This review outlines the growing relevance of supramolecular hydrogels in biosensing and bioassay devices, highlighting recent advancements that deliver increased sensitivity, real-time monitoring, and multiplexing capabilities through the distinctive properties of these nanomaterials. Furthermore, the exploration extends to additional applications, such as using hydrogels as three-dimensional matrices for cell-based assays.
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Bioensaio , Técnicas Biossensoriais , Hidrogéis , Hidrogéis/química , Técnicas Biossensoriais/métodos , Humanos , Bioensaio/métodos , Nanoestruturas/químicaRESUMO
Out-of-equilibrium self-assembly of metal nanoparticles (NPs) has been devised using different types of strategies and fuels, but achieving finite 3D structures with a controlled morphology through this assembly mode is still rare. Here, a spherical peptide-gold superstructure (PAuSS) is used as a template to control the out-of-equilibrium self-assembly of Au NPs, obtaining a transient 3D-branched Au-nanoshell (BAuNS) stabilized by sodium dodecyl sulphate (SDS). The BAuNS dismantles upon SDS concentration gradient equilibration over time in the sample solution, leading to NPs disassembly and regression to PAuSS. Notably, BAuNS assembly and disassembly promotes temporary interparticle plasmonic coupling, leading to reversible and tunable changes of their plasmonic properties, a highly desirable behavior in the development of optoelectronic nanodevices.
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Self-assembling peptides are of huge interest for biological, medical and nanotechnological applications. The enormous chemical variety that is available from the 20 amino acids offers potentially unlimited peptide sequences, but it is currently an issue to predict their supramolecular behavior in a reliable and cheap way. Herein we report a computational method to screen and forecast the aqueous self-assembly propensity of amyloidogenic pentapeptides. This method was found also as an interesting tool to predict peptide crystallinity, which may be of interest for the development of peptide based drugs.
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BACKGROUND: Early detection and removal of bladder cancer in patients is crucial to prevent tumor recurrence and progression. Because current imaging techniques may fail to detect small lesions of in situ carcinomas, patients with bladder cancer often relapse after initial diagnosis, thereby requiring frequent follow-up and treatments. RESULTS: In an attempt to obtain a sensitive and high-resolution imaging modality for bladder cancer, we have developed a photoacoustic imaging approach based on the use of PEGylated gold nanorods (GNRs) as a contrast agent, functionalized with the peptide cyclic [CphgisoDGRG] (Iso4), a selective ligand of α5ß1 integrin expressed by bladder cancer cells. This product (called GNRs@PEG-Iso4) was produced by a simple two-step procedure based on GNRs activation with lipoic acid-polyethyleneglycol(PEG-5KDa)-maleimide and functionalization with peptide Iso4. Biochemical and biological studies showed that GNRs@PEG-Iso4 can efficiently recognize purified integrin α5ß1 and α5ß1-positive bladder cancer cells. GNRs@PEG-Iso4 was stable and did not aggregate in urine or in 5% sodium chloride, or after freeze/thaw cycles or prolonged exposure to 55 °C, and, even more importantly, do not settle after instillation into the bladder. Intravesical instillation of GNRs@PEG-Iso4 into mice bearing orthotopic MB49-Luc bladder tumors, followed by photoacoustic imaging, efficiently detected small cancer lesions. The binding to tumor lesions was competed by a neutralizing anti-α5ß1 integrin antibody; furthermore, no binding was observed to healthy bladders (α5ß1-negative), pointing to a specific targeting mechanism. CONCLUSION: GNRs@PEG-Iso4 represents a simple and robust contrast agent for photoacoustic imaging and diagnosis of small bladder cancer lesions.
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Nanotubos , Técnicas Fotoacústicas , Neoplasias da Bexiga Urinária , Animais , Camundongos , Meios de Contraste , Integrina alfa5beta1 , Neoplasias da Bexiga Urinária/diagnóstico por imagem , OuroRESUMO
Bromination is herein exploited to promote the emergence of elastic behavior in a short peptide-SDSYGAP-derived from resilin, a rubber-like protein exerting its role in the jumping and flight systems of insects. Elastic and resilient hydrogels are obtained, which also show self-healing behavior, thanks to the promoted non-covalent interactions that limit deformations and contribute to the structural recovery of the peptide-based hydrogel. In particular, halogen bonds may stabilize the ß-sheet organization working as non-covalent cross-links between nearby peptide strands. Importantly, the unmodified peptide (i.e., wild type) does not show such properties. Thus, SDSY(3,5-Br)GAP is a novel minimalist peptide elastomer.
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Drosophila melanogaster , Halogenação , Animais , Drosophila melanogaster/metabolismo , Elasticidade , Hidrogéis , Proteínas de Insetos , Peptídeos/químicaRESUMO
Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
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Hexoquinase , Neoplasias , Animais , Morte Celular , Retículo Endoplasmático/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Camundongos , Mitocôndrias , Membranas Mitocondriais/metabolismo , Neoplasias/metabolismoRESUMO
Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs' cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.
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Aloe , Antineoplásicos , Produtos Biológicos , Neoplasias da Mama , Emodina , Aloe/química , Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Apoptose , Produtos Biológicos/farmacologia , Emodina/farmacologia , Feminino , Humanos , Peptídeos/farmacologiaRESUMO
All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.
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Núcleo Celular/enzimologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , NAD/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Estresse Fisiológico , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Cinética , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Células NIH 3T3 , Mononucleotídeo de Nicotinamida/química , Nicotinamida Fosforribosiltransferase/química , Ligação Proteica , Multimerização Proteica , Transporte ProteicoRESUMO
Extracellular HMGB1 triggers inflammation following infection or injury and supports tumorigenesis in inflammation-related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide-containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, diflunisal does not inhibit TLR4-dependent responses. Our findings clarify the mode of action of diflunisal and open the way to the rational design of functionally specific anti-inflammatory drugs.
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Quimiocina CXCL12/metabolismo , Diflunisal/farmacologia , Proteína HMGB1/metabolismo , Leucócitos/metabolismo , Células 3T3 , Animais , Quimiotaxia/efeitos dos fármacos , Diflunisal/química , Dissulfetos/metabolismo , Ácido Glicirrízico/farmacologia , Humanos , Inflamação/patologia , Leucócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Espectroscopia de Ressonância Magnética , CamundongosRESUMO
BACKGROUND: Gold nanospheres tagged with peptides containing isoDGR (isoAsp-Gly-Arg), an αvß3 integrin binding motif, represent efficient carriers for delivering pro-inflammatory cytokines to the tumor vasculature. We prepared bi- or trifunctional nanoparticles bearing tumor necrosis factor-α (TNF) and/or interleukin-12 (IL12) plus a peptide containing isoDGR, and we tested their anti-cancer effects, alone or in combination with doxorubicin, in tumor-bearing mice. RESULTS: In vitro biochemical studies showed that both nanodrugs were monodispersed and functional in terms of binding to TNF and IL12 receptors and to αvß3. In vivo studies performed in a murine model of fibrosarcoma showed that low doses of bifunctional nanoparticles bearing isoDGR and TNF (corresponding to few nanoparticles per cell) delayed tumor growth and increased the efficacy of doxorubicin without worsening its toxicity. Similar effects were obtained using trifunctional nanoparticles loaded with isoDGR, TNF and IL12. Mechanistic studies showed that nanoparticles bearing isoDGR and TNF could increase doxorubicin penetration in tumors a few hours after injection and caused vascular damage at later time points. CONCLUSION: IsoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multi-cytokine delivery to cells of the tumor vasculature. Extremely low doses of isoDGR-coated nanodrugs functionalized with TNF or TNF plus IL12 can enhance doxorubicin anti-tumor activity.
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Antineoplásicos/farmacologia , Citocinas , Doxorrubicina/farmacologia , Nanoestruturas/química , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Integrina alfaVbeta3 , Interleucina-12 , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/uso terapêutico , Fator de Necrose Tumoral alfaRESUMO
The honeybee, Apis mellifera L. (Hymenoptera: Apidae), a keystone pollinator of wild plant species and agricultural crops, is disappearing globally due to parasites and diseases, habitat loss, genetic constraints, beekeeper management issues and to the widespread use of pesticides. Besides insecticides, widely studied in this species, honeybees are also exposed to herbicides and fungicides and heavy metals whose lethal and sublethal effects need to be investigated. In this context, our study aimed to evaluate the effects of fungicides and of heavy metals on honeybees and to develop and apply a multi-biomarker approach that include an Integrated Biological Index (IBRv2) to assess the toxicological status of this species. Biomarkers of neurotoxicity (AChE and CaE), metabolic alteration (ALP, and GST) and immune system (LYS, granulocytes) were measured, following honeybees' exposure to cadmium or to a crop fungicide, using the genotoxic compound EMS as positive control. A biomarker of genotoxicity (NA assay) was developed and applied for the first time in honeybees. At the doses tested, all the contaminants showed sublethal toxicity to the bees, highlighting in particular genotoxic effects. The data collected were analyzed by an IBRv2 index, which integrated the seven biomarkers used in this study. IBRv2 index increased with increasing cadmium or fungicide concentrations. The IBRv2 represents a simple tool for a general description of honeybees ecotoxicological health status. Results highlight the need for more in-depth investigations on the effects of fungicides on non-target organisms, such as honeybees, using sensitive methods for the determination of sublethal effects. This study contributes to the development of a multi-biomarker approach to be used for a more accurate ecotoxicological environmental monitoring of these animals.
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Abelhas/efeitos dos fármacos , Biomarcadores/análise , Fungicidas Industriais/toxicidade , Metais Pesados/toxicidade , Testes de Toxicidade/métodos , Animais , Testes de Mutagenicidade/métodosRESUMO
Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein-protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.
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Carcinógenos/química , Proteínas de Ciclo Celular/química , Chaperoninas/química , Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Carcinógenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Dobramento de ProteínaRESUMO
Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. A second putative FGF2 binding sequence was also identified in repeat 11C of both TSPs. Computational docking analysis predicted that both the TSP-2 and TSP-1-derived heptapeptides interacted with FGF2 with comparable binding properties. Accordingly, small molecules based on the TSP-1 active sequence blocked TSP-2/FGF2 interaction. Binding of TSP-2 to FGF2 impaired the growth factor ability to interact with its cellular receptors, since TSP-2-derived fragments prevented the binding of FGF2 to both heparin (used as a structural analog of heparan sulfate proteoglycans) and FGFR-1. These findings identify TSP-2 as a new FGF2 ligand that shares with TSP-1 the same molecular requirements for interaction with the growth factor and a comparable capacity to block FGF2 interaction with proangiogenic receptors. These features likely contribute to TSP-2 antiangiogenic and antineoplastic activity, providing the rationale for future therapeutic applications.
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Fator 2 de Crescimento de Fibroblastos/química , Ressonância de Plasmônio de Superfície , Trombospondinas/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos , Sequências Repetitivas de Aminoácidos , Trombospondinas/metabolismoRESUMO
Although the traditional strategy of developing general medical treatments for heterogeneous patient populations has a well-established track record, the acknowledgment that one-size-does-not-fit-all is pushing health-care to enter a new era of tailored interventions. The advent of precision medicine is fueled by the high-throughput analysis of individual DNA variants and mRNA expression profiles. However, due to the role of proteins in providing a more direct view of disease states than genomics alone, the ability to comprehensively analyze protein alterations and post translational modifications (PTMs) is a necessary step to unravel disease mechanisms, develop novel biomarkers and targeted therapies. Protein and peptide microarrays can play a major role in this frame, due to high-throughput, low sample consumption and wide applicability. Here, their current role and potentialities are discussed through the review of some promising applications in the fields of PTMs analysis, enzyme screening, high-content immune-profiling and the phenotyping of extracellular vesicles.
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Peptídeos/metabolismo , Medicina de Precisão/métodos , Análise Serial de Proteínas/métodos , Bioensaio , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Peptídeos/químicaRESUMO
The mosquito-borne viral disease caused by the Dengue virus is an expanding global threat. Diagnosis in low-resource-settings and epidemiological surveillance urgently requires new immunoprobes for serological tests. Structure-based epitope prediction is an efficient method to design diagnostic peptidic probes able to reveal specific antibodies elicited in response to infections in patients' sera. In this study, we focused on the Dengue viral envelope protein (E); computational analyses ranging from extensive Molecular Dynamics (MD) simulations and energy-decomposition-based prediction of potentially immunoreactive regions identified putative epitope sequences. Interestingly, one such epitope showed internal dynamic and energetic properties markedly different from those of other predicted sequences. The epitope was thus synthesized as a linear peptide, modified for chemoselective immobilization on microarrays and used in a serological assay to discriminate Dengue-infected individuals from healthy controls. The synthetic epitope probe showed a diagnostic performance comparable to that of the full antigen in terms of specificity and sensitivity. Given the high level of sequence identity among different flaviviruses, the epitope was immune-reactive towards Zika-infected sera as well. The results are discussed in the context of the quest for new possible structure-dynamics-based rules for the prediction of the immunoreactivity of selected antigenic regions with potential pan-flavivirus immunodiagnostic capacity.
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Vírus da Dengue/imunologia , Dengue/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais , Biologia Computacional , Reações Cruzadas/imunologia , Dengue/sangue , Dengue/virologia , Vírus da Dengue/patogenicidade , Mapeamento de Epitopos , Humanos , Simulação de Dinâmica Molecular , Peptídeos/imunologia , Zika virus/imunologia , Zika virus/patogenicidade , Infecção por Zika virus/sangue , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.
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Antineoplásicos/uso terapêutico , Nanopartículas/química , Neuroblastoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Humanos , Neuroblastoma/metabolismo , Neuropilina-1/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies. DESIGN: We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. RESULTS: Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+ T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo. CONCLUSIONS: These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.
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Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , DNA Complementar/análise , Epitopos de Linfócito T/genética , Proteína da Polipose Adenomatosa do Colo/genética , Proteínas de Ciclo Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Epitopos de Linfócito T/imunologia , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Expressão Gênica , Antígenos HLA/genética , Antígenos HLA/imunologia , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Neoplásicas/imunologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Smad4/genética , Proteína Smad4/imunologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.
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The activity profile of many biologically relevant proteins and peptides often relies on a precise 3D structural organization. In this context, disulfide bonds are natural covalent constraints that play a key role in driving and stabilizing the folding pattern of these molecules. Despite its prominent significance as structural motif, the disulfide bond itself is inherently unstable under physiological conditions, posing a major limit to the use and development of disulfide-rich peptides and proteins as molecular tools and drug lead compounds. To tackle this restriction, disulfide engineering with stable functional analogues has arisen a considerable interest. Here, the most popular approaches to disulfide replacement are reviewed and discussed with particular emphasis on advantages and limitations under both functional and synthetic perspectives.