RESUMEN
A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1-13) potently inhibits the Nln activity while dynorphin A (1-8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.
Asunto(s)
Dinorfinas , Neurotensina , Humanos , Especificidad por Sustrato , Dinorfinas/química , Dinorfinas/metabolismo , Neurotensina/química , Neurotensina/metabolismo , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/antagonistas & inhibidores , Unión Proteica , Cristalografía por Rayos X , Modelos Moleculares , Dominio Catalítico , Bradiquinina/química , Bradiquinina/metabolismo , Angiotensinas/metabolismo , Angiotensinas/química , Secuencia de AminoácidosRESUMEN
The development of multitarget opioid drugs has emerged as an attractive approach for innovative pain management with reduced side effects. In the present study, a novel hybrid peptide BNT12 containing the opioid and neurotensin (NT)-like fragments was synthesized and pharmacologically characterized. In acute radiant heat paw withdrawal test, intracerebroventricular (i.c.v.) administration of BNT12 produced potent antinociception in mice. The central antinociceptive activity of BNT12 was mainly mediated by µ-, δ-opioid receptor, neurotensin receptor type 1 (NTSR1) and 2 (NTSR2), supporting a multifunctional agonism of BNT12 in the functional assays. BNT12 also exhibited significant antinociceptive effects in spared nerve injury (SNI)-neuropathic pain, complete Freund's adjuvant (CFA)-induced inflammatory pain, acetic acid-induced visceral and formalin-induced pain after i.c.v. administration. Furthermore, BNT12 exhibited substantial reduction of acute antinociceptive tolerance, shifted the dose-response curve to the right by only 1.3-fold. It is noteworthy that BNT12 showed insignificant chronic antinociceptive tolerance at the supraspinal level. In addition, BNT12 exhibited reduced or no opioid-like side effects on conditioned place preference (CPP) response, naloxone-precipitated withdrawal response, acute hyperlocomotion, motor coordination, gastrointestinal transit, and cardiovascular responses. The present investigation demonstrated that the novel hybrid peptide BNT12 might serve as a promising analgesic candidate with limited opioid-like side effects.
Asunto(s)
Neurotensina , Receptores de Neurotensina , Animales , Masculino , Ratones , Neurotensina/análogos & derivados , Neurotensina/farmacología , Neurotensina/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/agonistas , Analgésicos/farmacología , Analgésicos/química , Analgésicos/administración & dosificación , Analgésicos Opioides/farmacología , Analgésicos Opioides/administración & dosificación , Tolerancia a Medicamentos , Dolor/tratamiento farmacológicoRESUMEN
Neurotensin (NT) is a 13-residue endogenous peptide found in mammals, with neurotransmission and hormonal roles in the central nervous system and gastrointestinal tract, respectively. The first residue of NT is a pyroglutamate (pGlu) that makes the expression and purification of large amounts of NT with native modification challenging. Here, we describe a simple and efficient procedure for expression and purification of large amounts of NT based on using the small ubiquitin-like modifier (SUMO) as a fusion partner and subsequent enzymatic conversion of the N-terminal glutamine to pGlu. Yields of 13 mg/L and 8 mg/L of pure peptide were obtained from expression in rich and minimal media, respectively. The method is adaptable to expression and purification of proteins and peptides with pGlu modification in a wide range of eukaryotic and prokaryotic expression hosts.
Asunto(s)
Neurotensina , Ácido Pirrolidona Carboxílico , Animales , Neurotensina/genética , Neurotensina/química , Neurotensina/metabolismo , Péptidos/química , Glutamina , MamíferosRESUMEN
G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Unión al GTP Heterotriméricas/ultraestructura , Membrana Dobles de Lípidos , Nanoestructuras/química , Receptores de Neurotensina/metabolismo , Receptores de Neurotensina/ultraestructura , Regulación Alostérica , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/ultraestructura , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/ultraestructura , Guanosina Difosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Micelas , Modelos Moleculares , Neurotensina/química , Neurotensina/metabolismo , Conformación Proteica , Receptores de Neurotensina/química , Transducción de SeñalRESUMEN
The present study aimed to determine the primary sequence of ovine xenin and clarify the mRNA expression and peptide localization of xenin in the gastrointestinal tract in sheep. The colocalization of xenin and glucose-dependent insulinotropic polypeptide was also compared in the antrum and duodenum. Analysis of the nucleotide sequence of ovine xenin revealed a high degree (97.9%) of sequence homology of the sequence between sheep and cattle, and the amino acids sequence determined for ovine xenin coincided (100%) with that of other mammalian species. Real-time quantitative PCR for ovine xenin did not show regional difference in the mRNA expression ratio of xenin. In contrast to the real-time quantitative PCR results, anti-xenin positive cells were abundantly localized in the abomasal antrum (P < 0.01) and at a lesser amount in the duodenum, but no antixenin positive cells were observed in the other regions. Anti-xenin single-positive cells were in a majority in the abomasal antrum, whereas anti-xenin single-positive cells, and anti-GIP single-positive cells, and double-positive cells were even colocalized in the duodenum. These results suggest that abomasal antrum is a major source of xenin in the ovine gastrointestinal tract.
Asunto(s)
Tracto Gastrointestinal/química , Tracto Gastrointestinal/metabolismo , Expresión Génica , Neurotensina/genética , ARN Mensajero/análisis , Ovinos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Polipéptido Inhibidor Gástrico/análisis , Inmunohistoquímica , Masculino , Neurotensina/químicaRESUMEN
BACKGROUND: The overexpression of neurotensin subtype 1 receptors (NTS1Rs) in human tumors may be elegantly exploited for directing neurotensin (NT)-based radionuclide carriers specifically to cancer sites for theranostic purposes. We have recently shown that [99mTc]Tc-DT1 ([99mTc]Tc-[N4-Gly7]NT(7-13)) and [99mTc]Tc-DT5 ([99mTc]Tc-[N4-ßAla7,Dab9]NT(7-13)) show notably improved uptake in human colon adenocarcinoma WiDr xenografts in mice treated with neprilysin (NEP) inhibitors and/or angiotensin-converting enzyme (ACE) inhibitors compared with untreated controls. Aiming toward translation of this promising approach in NTS1R-positive pancreatic ductal adenocarcinoma (PDAC) patients, we now report on the impact of registered NEP/ACE inhibitors on the performance of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 in pancreatic cancer models. METHODS: The cellular uptake of [99mTc]Tc-DT1 and [99mTc]Tc-DT5 was tested in a panel of pancreatic cell lines, and their stability was assessed in mice treated or not treated with Entresto, lisinopril, or their combinations. Biodistribution was conducted in severe combined immunodeficiency (SCID) mice bearing pancreatic AsPC-1 xenografts. RESULTS: The Entresto + lisinopril combination maximized the metabolic stability of the fast-internalizing [99mTc]Tc-DT1 in mice, resulting in notably enhanced tumor uptake (7.05 ± 0.80% injected activity (IA)/g vs. 1.25 ± 0.80% IA/g in non-treated controls at 4 h post-injection; p < 0.0001). CONCLUSIONS: This study has shown the feasibility of optimizing the uptake of [99mTc]Tc-DT1 in pancreatic cancer models with the aid of clinically established NEP/ACE inhibitors, in favor of clinical translation prospects.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Neurotensina/química , Compuestos de Organotecnecio/química , Neoplasias Pancreáticas/metabolismo , Péptidos/administración & dosificación , Inhibidores de Proteasas/administración & dosificación , Receptores de Neurotensina/metabolismo , Aminobutiratos/administración & dosificación , Aminobutiratos/farmacología , Animales , Compuestos de Bifenilo , Línea Celular Tumoral , Combinación de Medicamentos , Estabilidad de Medicamentos , Humanos , Lisinopril/administración & dosificación , Lisinopril/farmacología , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Péptidos/química , Péptidos/farmacocinética , Inhibidores de Proteasas/farmacología , Tetrazoles/administración & dosificación , Tetrazoles/farmacología , Distribución Tisular , ValsartánRESUMEN
The natural tridecapeptide neurotensin has been emerged as a promising therapeutic scaffold for the treatment of neurological diseases and cancer. In this work, we aimed to identify the top 100 most cited original research papers as well as recent key studies related to neurotensins. The Web of Science Core Collection database was searched and the retrieved research articles were analyzed by using the VOSviewer software. The most cited original articles were published between 1973 and 2013. The top-cited article was by Carraway and Leeman reporting the discovery of neurotensin in 1973. The highly cited terms were associated with hypotension and angiotensin-converting-enzyme. The conducted analysis reveals the therapeutic potentials of neurotensin, and further impactful research toward its clinical development is warrantied.
Asunto(s)
Antineoplásicos/uso terapéutico , Fármacos del Sistema Nervioso Central/uso terapéutico , Neoplasias/tratamiento farmacológico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Neurotensina/uso terapéutico , Animales , Antineoplásicos/química , Fármacos del Sistema Nervioso Central/química , Humanos , Neurotensina/químicaRESUMEN
Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.
Asunto(s)
Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Neurotensina/química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinética , Animales , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Morfolinos/administración & dosificación , Morfolinos/química , Morfolinos/farmacocinética , Oligonucleótidos Antisentido/químicaRESUMEN
Recent advances and large-scale use of hybrid imaging modalities like PET-CT have led to the necessity of improving nano-drug carriers that can facilitate both functional and metabolic screening in nuclear medicine applications. In this study, we focused on the evaluation of four potential imaging nanoparticle structures labelled with the 68Ga positron emitter. For this purpose, we functionalized NHS-activated PEG-gold nanoparticles with 68Ga-DOTA-Neuromedin B, 68Ga-DOTA-PEG(4)-BBN(7-14), 68Ga-DOTA-NT and 68Ga-DOTA-Neuromedin N. In vitro binding kinetics and specific binding to human HT-29 colon carcinoma cells and DU-145 prostate carcinoma cells respectively were assessed, over 75% retention being obtained in the case of 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP in prostate tumour cells and over 50% in colon carcinoma cells. Biodistribution in NU/J mice highlighted a three-fold uptake increase in tumours at 30 min post-injection of 68Ga-DOTA-NT-AuNP and 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP compared to 68Ga-DOTA-NT and 68Ga-DOTA-PEG(4)-BBN(7-14) respectively, therewith fast distribution in prostate and colon tumours and minimum accumulation in non-targeted tissues.
Asunto(s)
Neoplasias del Colon/diagnóstico por imagen , Oro/administración & dosificación , Neuroquinina B/análogos & derivados , Neurotensina/química , Fragmentos de Péptidos/química , Péptidos/química , Neoplasias de la Próstata/diagnóstico por imagen , Animales , Disponibilidad Biológica , Bombesina/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Radioisótopos de Galio/administración & dosificación , Radioisótopos de Galio/química , Radioisótopos de Galio/farmacocinética , Oro/química , Oro/farmacocinética , Células HT29 , Humanos , Masculino , Nanopartículas del Metal/química , Ratones , Trasplante de Neoplasias , Neuroquinina B/química , Péptidos/administración & dosificación , Péptidos/farmacocinética , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/metabolismo , Receptores de Bombesina/metabolismo , Receptores de Neurotensina/metabolismoRESUMEN
The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Péptidos/farmacología , Animales , Antígenos CD/química , Antígenos CD/efectos de los fármacos , Antígenos CD/genética , Antígenos CD/farmacología , Sistema Nervioso Central/efectos de los fármacos , Cistina/química , Cistina/genética , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Ratones , Neuropéptidos/química , Neuropéptidos/farmacología , Neurotensina/química , Neurotensina/farmacología , Péptidos/química , Unión Proteica/efectos de los fármacos , Receptores de Transferrina/química , Receptores de Transferrina/efectos de los fármacos , Receptores de Transferrina/genéticaRESUMEN
BACKGROUND: Cancer is the leading cause of death worldwide. Early detection can reduce the disadvantageous effects of diseases and the mortality in cancer. Nuclear medicine is a powerful tool that has the ability to diagnose malignancy without harming normal tissues. In recent years, radiolabeled peptides have been investigated as potent agents for cancer detection. Therefore, it is necessary to modify radiopeptides in order to achieve more effective agents. OBJECTIVE: This review describes modifications in the structure of radioconjugates with spacers who have improved the specificity and sensitivity of the peptides that are used in oncologic diagnosis and therapy. METHODS: To improve the biological activity, researchers have conjugated these peptide analogs to different spacers and bifunctional chelators. Many spacers of different kinds, such as hydrocarbon chain, amino acid sequence, and poly (ethyleneglycol) were introduced in order to modify the pharmacokinetic properties of these biomolecules. RESULTS: Different spacers have been applied to develop radiolabeled peptides as potential tracers in nuclear medicine. Spacers with different charge and hydrophilicity affect the characteristics of peptide conjugate. For example, the complex with uncharged and hydrophobic spacers leads to increased liver uptake, while the composition with positively charged spacers results in high kidney retention. Therefore, the pharmacokinetics of radio complexes correlates to the structure and total charge of the conjugates. CONCLUSION: Radio imaging technology has been successfully applied to detect a tumor in the earliest stage. For this purpose, the assessment of useful agents to diagnose the lesion is necessary. Developing peptide radiopharmaceuticals using spacers can improve in vitro and in vivo behavior of radiotracers leading to better noninvasive detection and monitoring of tumor growth.
Asunto(s)
Neoplasias/diagnóstico , Neoplasias/radioterapia , Péptidos/química , Péptidos/uso terapéutico , Radiofármacos/química , Radiofármacos/uso terapéutico , Animales , Bombesina/química , Bombesina/uso terapéutico , Gastrinas/química , Gastrinas/uso terapéutico , Humanos , Hormonas Estimuladoras de los Melanocitos/química , Hormonas Estimuladoras de los Melanocitos/uso terapéutico , Neurotensina/química , Neurotensina/uso terapéutico , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Somatostatina/química , Somatostatina/uso terapéuticoRESUMEN
Ligand-receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand-receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand-receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT-NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Neurotensina , Receptores de Neurotensina , Ligandos , Modelos Moleculares , Neurotensina/química , Polifenoles , Receptores de Neurotensina/químicaRESUMEN
We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.
Asunto(s)
Neoplasias Colorrectales/enzimología , Metaloendopeptidasas/análisis , Proteoma/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Cromatografía Liquida , Electroforesis , Humanos , Metaloendopeptidasas/química , Neurotensina/química , Biblioteca de Péptidos , Espectrometría de Masas en TándemRESUMEN
Xenin-25 undergoes rapid enzyme metabolism following secretion. Early studies demonstrated bioactivity of a C-terminal hexapeptide fragment of xenin-25, namely xenin-6, which were enhanced through introduction of a reduced N-terminal peptide bond, to yield Ψ-xenin-6. The present study was undertaken to define the biological actions and potential antidiabetic properties of Ψ-xenin-6. In vitro enzymatic stability, insulin and glucagon secretory activity, as well as effects on beta-cell survival were determined. Studies in mice were used to assess the impact of Ψ-xenin-6 on glucose homeostasis and satiety. Ψ-xenin-6 was resistant to murine plasma degradation. In BRIN-BD11â¯cells and isolated murine islets, Ψ-xenin-6 significantly stimulated insulin secretion, and prominently enhanced the insulinotropic actions of GIP. Xenin-6 and Ψ-xenin-6 had no impact on glucagon secretion, although xenin-6 partially reversed the glucagonotropic action of GIP. Further in vitro investigations revealed that, similar to GLP-1, Ψ-xenin-6 significantly augmented proliferation of human and rodent clonal beta-cells, whilst also fully protecting against cytokine-induced beta-cell cytotoxicity, with greater potency than xenin-25 and xenin-6. When administered to mice in combination with glucose, Ψ-xenin-6 significantly reduced glucose levels and enhanced glucose-induced insulin release, with a duration of biological action beyond 8â¯h. Ψ-xenin-6 also significantly enhanced the glucose-lowering action of GIP in vivo. In overnight fasted mice, Ψ-xenin-6 exhibited satiety actions at both 25 and 250â¯nmol/kg. These data demonstrates that Ψ-xenin-6 is a metabolically stable C-terminal fragment analogue of xenin-25, with a metabolic action profile that merits further study as a potential antidiabetic compound.
Asunto(s)
Glucagón/metabolismo , Hipoglucemiantes , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Neurotensina , Animales , Línea Celular , Glucosa/farmacología , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Ratones , Neurotensina/química , Neurotensina/farmacologíaRESUMEN
Crystal structures of neurotensin receptor subtype 1 (NTS1) allowed us to visualize the binding mode of the endogenous peptide hormone neurotensin and its pharmacologically active C-terminal fragment NT(8-13) within the orthosteric binding pocket of NTS1. Beneath the orthosteric binding pocket, we detected a cavity that exhibits different sequences in the neurotensin receptor subtypes NTS1 and NTS2. In this study, we explored this allosteric binding pocket using bitopic test peptides of type NT(8-13)-Xaa, in which the C-terminal part of NT(8-13) is connected to different amino acids that extend into the newly discovered pocket. Our test compounds showed nanomolar affinities for NTS1, a measurable increase in subtype selectivity compared to the parent peptide NT(8-13), and the capacity to activate the receptor in an IP accumulation assay. Computational investigation of the selected test compounds at NTS1 showed a conserved binding mode within the orthosteric binding pocket, whereas the allosteric cavity was able to adapt to different residues, which suggests a high degree of structural plasticity within that cavity of NTS1.
Asunto(s)
Sitio Alostérico , Sitios de Unión , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neurotensina/química , Fragmentos de Péptidos/química , Receptores de Neurotensina/química , Regulación Alostérica , Animales , Línea Celular , Humanos , Ligandos , Neurotensina/metabolismo , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Receptores de Neurotensina/metabolismoRESUMEN
Neurotensin (NT) (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) exerts a dual function as a neurotransmitter/neuromodulator in the central nervous system and as a hormone/cellular mediator in periphery. This dual function of NT establishes a connection between brain and peripheral tissues that renders this peptide a central player in energy homeostasis. Many biological actions of NT are mediated through its interaction with three types of NT receptors (NTS receptors). Despite its role in energy homeostasis, NT has a short half-life that hampers further determination of the biological actions of this peptide and its receptors in brain and periphery. The short half-life of NT is due to the proteolytic degradation of its C-terminal side by several endopeptidases. Therefore, it is important to synthesize NT analogues with resistant bonds against metabolic deactivation. Based on these findings, we herein report the synthesis of ten linear, two cyclic and two dimeric analogues of NT with modifications in its structure that improve their metabolic stability, while retaining the ability to bind to NTS receptors. Modifications at position 11 (introduction of D-Tyrosine (OEthyl) [D-Tyr(Et)] or D-1-naphtylalanine [D-1-Nal] were combined with introduction of a L-Lysine or a D-Arginine at positions 8 or 9, and 1-[2-(aminophenyl)-2-oxoethyl]-1H-pyrrole-2-carboxylic acid (AOPC) at positions 7 or 8, resulting in compounds NT4-NT21. AOPC is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetic compounds. To biologically evaluate these analogues, we determined their plasma stability and their binding affinities to type 1 NT receptor (NTS1), endogenously expressed in HT-29 cells, Among the fourteen NT analogues, compounds, NT5, NT6, and NT8, which have D-Tyr(Et) at position 11, bound to NTS1 in a dose-response manner and with relatively high affinity but still lower than that of the natural peptide. Despite their lower binding affinities compared to NT, the NT5, NT6, and NT8 exhibited a remarkably higher stability, as a result of their chemistry, which provides protection from enzymatic activity. These results will set the basis for the rational design of novel NT molecules with improved pharmacological properties and enhanced enzymatic stability.
Asunto(s)
Aminoácidos/química , Neurotensina/química , Peptidomiméticos/síntesis química , Peptidomiméticos/metabolismo , Secuencia de Aminoácidos , Técnicas de Química Sintética , Cromatografía Líquida de Alta Presión , Células HT29 , Humanos , Espectrometría de Masas , Modelos Moleculares , Simulación de Dinámica Molecular , Peptidomiméticos/farmacología , Receptores de Neurotensina/químicaRESUMEN
Sortilin is a multifunctional neuronal receptor involved in sorting of neurotrophic factors and apoptosis signaling. So far, structural characterization of sortilin and its endogenous ligands has been limited to crystallographic studies of sortilin in complex with the neuropeptide neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of sortilin to binding biological ligands including the peptides neurotensin and the sortilin propeptide and the proteins progranulin and pro-nerve growth factor-ß. The results show that the ligands use two binding sites inside the cavity of the ß-propeller of sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of sortilin. Our findings improve our structural understanding of sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.
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Proteínas Adaptadoras del Transporte Vesicular/química , Factor de Crecimiento Nervioso/química , Neurotensina/química , Progranulinas/química , Precursores de Proteínas/química , Proteínas Recombinantes de Fusión/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Ligandos , Modelos Moleculares , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Neurotensina/genética , Neurotensina/metabolismo , Progranulinas/genética , Progranulinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Optogenetics is a recently established neuromodulation technique in which photostimulation is used to manipulate neurons with high temporal and spatial precision. However, sequential genetic and optical insertion with double brain implantation tends to cause excessive tissue damage. In addition, the incorporation of light-sensitive genes requires the utilization of viral vectors, which remains a safety concern. Here, by combining device fabrication design, nanotechnology, and cell targeting technology, we developed a new gene-embedded optoelectrode array for neural implantation to enable spatiotemporal electroporation (EP) for gene delivery/transfection, photomodulation, and synchronous electrical monitoring of neural signals in the brain via one-time implantation. A biotic-abiotic neural interface (called PG) composed of reduced graphene oxide and conductive polyelectrolyte 3,4-ethylenedioxythiophene-modified amphiphilic chitosan was developed to form a nanostructural hydrogel with assembled nanodomains for encapsulating nonviral gene vectors (called PEI-NT-pDNA) formulated by neurotensin (NT) and polyethylenimine (PEI)-coupled plasmid DNA (pDNA). The PG can maintain high charge storage ability to respond to a minimal current of 125 µA for controllable gene delivery. The in vitro analysis of PG-PEI-NT-pDNA on the microelectrode array chip showed that the microelectrodes provided electrically inductive electropermeabilization, which permitted gene transfection into localized rat adrenal pheochromocytoma cells with a strong green fluorescent protein expression that was up to 8-fold higher than that in nontreated cells. Furthermore, the in vivo implantation enabled on-demand spatiotemporal gene transfection to neurons with 10-fold enhancement of targeting ability compared with astrocytes. Finally, using the real optogenetic opsin channelrhodopsin-2, the flexible neural probe incorporated with an optical waveguide fiber displayed photoevoked extracellular spikes in the thalamic ventrobasal region after focal EP for only 7 days, which provided a proof of concept for the use of photomodulation to facilitate neural therapies.
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Nanoestructuras/química , Neuronas/fisiología , Optogenética/métodos , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Supervivencia Celular/efectos de los fármacos , Conductividad Eléctrica , Electrodos Implantados , Grafito/química , Hidrogeles/química , Microelectrodos , Microscopía Fluorescente , Neurotensina/química , Células PC12 , Plásmidos/química , Plásmidos/metabolismo , Polietileneimina/química , Polímeros/química , Ratas , TransfecciónRESUMEN
Efficient drug accumulation in tumor cells is essential for cancer therapy. Herein, we developed dimeric prodrug self-delivery nanoparticles (NPs) with enhanced drug loading and bioreduction responsiveness for triple negative breast cancer (TNBC) therapy. Specially designed camptothecin dimeric prodrug (CPTD) containing a disulfide bond was constructed to realize intracellular redox potential controlled drug release. Direct conjugation of hydrophobic CPTD to poly(ethylene glycol) PEG5000, a prodrug-based amphiphilic CPTD-PEG5000 co-polymer was synthesized, which could encapsulate parental CPTD prodrug spontaneously and form ultrastable NPs due to the highly analogous structure. Such dimeric prodrug self-delivery nanoparticles showed ultrahigh stability with critical micelle concentration as low as 0.75 µg/mL and remained intact during endocytosis. In addition, neurotensin (NT), a 13 amino acid ligand, was further modified on the nanoparticles for triple negative breast cancer (TNBC) targeting. Optimized NT-CPTD NPs showed improved pharmacokinetics profile and increased drug accumulation in TNBC lesions than free CPT, which largely reduced the systemic toxicity and presented an improved anticancer efficacy in vivo. In summary, with advantages of extremely high drug loading capacity, tumor microenvironmental redox responsiveness, and targeted TNBC accumulation, NT-CPTD NPs showed their potential for effective triple negative breast cancer therapy.
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Portadores de Fármacos/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Apoptosis , Camptotecina/administración & dosificación , Línea Celular Tumoral , Dimerización , Ditiotreitol/química , Endocitosis , Femenino , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Ligandos , Ratones , Micelas , Neurotensina/química , Oxidación-Reducción , Polímeros/química , Profármacos/química , Temperatura , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
BACKGROUND: Neurotensin receptors are overexpressed in several cancer types including pancreatic ductal adenocarcinoma. Three NTR subtypes have been cloned: NTR-1, NTR-2 and NTR-3. The most expressed NTR-1 is not present in normal pancreatic tissue and has a low expression in chronic pancreatitis. OBJECTIVE: Objective of this study was to test in vitro affinity of the new 68Ga labelled neurotensin analogue DOTA-NT-20.3 (fragment 6-13, Ac-Lys(DOTA)-Pro-Arg(N-CH3)-Arg-Pro-Tyr-Tle-Leu) on the human pancreatic ductal adenocarcinoma cell line AsPC-1. METHOD: For the preparation of 68Ga-DOTA-NT-20.3, 68GaCl3 solution (eluted from 68Ge/68Ga generator) and 50 µg of precursor (Iason, Graz, Austria) water dissolved were used in an automatic synthesis module. The labeled compound was added to cell culture flask and incubated at 37°C. At various time points after tracer addition up to 80min, cells were recovered, rinsed and counted for radioactivity. Results were expressed as percent binding normalized to 200000 cells and affinity parameters were calculated. RESULTS: Labeling yield was ≥98 %. The molar ratio between labelled and total peptide was about 1/400. AsPC-1 cell line showed rapid uptake of the tracer including surface and internalized binding, tending to a plateau phase 80 min after tracer addition (11%/200.000 cells). The Kd (7.335 pmol) and Bmax (90.52 kBq) value indicated high tracer affinity for AsPC-1cell line especially if compared with the literature data regarding other malignancies (e.g. colonic cancer cell line). Binding sites were 1.09x106 sites per cell. CONCLUSION: New tracer 68Ga-DOTA-NT-20.3 can be a suitable candidate for the clinical use in patients with pancreatic ductal adenocarcinoma.