RESUMEN
The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr-Purcell-Meiboom-Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease-Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.
Asunto(s)
Proteínas Portadoras/farmacocinética , Proteasa del VIH/química , Proteasa del VIH/farmacocinética , VIH-1/enzimología , Espectroscopía de Resonancia Magnética/métodos , Dominios y Motivos de Interacción de Proteínas , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/farmacocinética , Secuencia de Aminoácidos , Fenómenos Biofísicos , Proteínas Portadoras/química , Dominio Catalítico , Farmacorresistencia Viral/genética , Proteasa del VIH/genética , VIH-1/genética , Cinética , Imagen por Resonancia Magnética , Modelos Moleculares , Mutagénesis , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteolisis , Proteínas Recombinantes , Especificidad por Sustrato , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Drug delivery into tumors and metastases is a major challenge in the eradication of cancers such as epithelial ovarian carcinoma. Cationic cell-penetrating peptides (CPPs) are a promising group of delivery vehicles to mediate cellular entry of molecules that otherwise poorly enter cells. However, little is known about their penetration behavior in tissues. Here, we investigated penetration of cationic CPPs in 3D ovarian cancer spheroids and patient-derived 3D tumor explants. Penetration kinetics and distribution after long-term incubation were imaged by confocal microscopy. In addition, spheroids and tumor explants were dissociated and cell-associated fluorescence determined by flow cytometry. CPPs with high uptake activity showed enhanced sequestration in the periphery of the spheroid, whereas less active CPPs were able to penetrate deeper into the tissue. CPPs consisting of d-amino acids were advantageous over l-amino acid CPPs as they showed less but long lasting cellular uptake activity, which benefitted penetration and retention over time. In primary tumor cultures, in contrast to nonaarginine, the amphipathic CPP penetratin was strongly sequestered by cell debris and matrix components pointing towards arginine-rich CPPs as a preferred choice. Overall, the data show that testing in 3D models leads to a different choice of the preferred peptide in comparison to a standard 2D cell culture.
Asunto(s)
Péptidos de Penetración Celular/farmacocinética , Vehículos Farmacéuticos/farmacocinética , Esferoides Celulares/efectos de los fármacos , Proteínas Portadoras/farmacocinética , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Cistadenocarcinoma Seroso/secundario , Femenino , Humanos , Microscopía Confocal , Neoplasias Ováricas/patología , Neoplasias Peritoneales/secundario , Vehículos Farmacéuticos/química , Relación Estructura-ActividadRESUMEN
The cell-penetrating peptide (CPP) penetratin has demonstrated potential as a carrier for transepithelial delivery of cargo peptides, such as the therapeutically relevant part of parathyroid hormone, i.e., PTH(1-34). The purpose of the present study was to elucidate the relevance of pH for PTH(1-34)-penetratin conjugates and coadministered penetratin with PTH(1-34) regarding transepithelial permeation of PTH(1-34) and cellular effects. Transepithelial permeation was assessed using monolayers of the Caco-2 cell culture model, and effects on Caco-2 cellular viability kinetics were evaluated by using the Real-Time-GLO assay as well as by microscopy following Tryphan blue staining. Morphological Caco-2 cell changes were studied exploiting the impedance-based xCELLigence system as well as optically using the oCelloscope setup. Finally, the effect of pH on the folding propensity of the PTH(1-34)-penetratin conjugate and its ability to disrupt lipid membranes were assessed by circular dichroism (CD) spectroscopy and the calcein release assay, respectively. The transepithelial PTH(1-34) permeation was not pH-dependent when applying the coadministration approach. However, by applying the conjugation approach, the PTH(1-34) permeation was significantly enhanced by lowering the pH from 7.4 to 5 but also associated with a compromised barrier and a lowering of the cellular viability. The negative effects on the cellular viability following cellular incubation with the PTH(1-34)-penetratin conjugate were moreover confirmed during real-time monitoring of the Caco-2 cell viability as well as by enhanced Tryphan blue uptake. In addition, morphological changes were primarily observed for cells incubated with the PTH(1-34)-penetratin conjugate at pH 5, which was moreover demonstrated to have an enhanced membrane permeating effect following lowering of the pH from 7.4 to 5. The latter observation was, however, not a result of better secondary folding propensity at pH 5 when compared to pH 7.4.
Asunto(s)
Proteínas Portadoras/química , Nanoconjugados/química , Hormona Paratiroidea/química , Hormona Paratiroidea/farmacocinética , Secuencia de Aminoácidos , Células CACO-2 , Proteínas Portadoras/farmacocinética , Permeabilidad de la Membrana Celular , Supervivencia Celular , Péptidos de Penetración Celular , Epitelio/metabolismo , Humanos , Concentración de Iones de Hidrógeno , PermeabilidadRESUMEN
Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 µmol/kg) and L1P3V8 (6 µmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.
Asunto(s)
Proteínas Portadoras/administración & dosificación , Trastornos Heredodegenerativos del Sistema Nervioso/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Péptidos/administración & dosificación , Péptidos/metabolismo , ARN Mensajero/antagonistas & inhibidores , Administración Intranasal , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Proteínas Portadoras/farmacocinética , Modelos Animales de Enfermedad , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada/métodos , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligopéptidos/farmacocinética , Péptidos/farmacocinética , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
To overcome the difficulty in delivery of biopharmaceuticals such as peptides and proteins to the brain, several approaches combining the ligands and antibodies targeting the blood-brain barrier (BBB) have been tried. However, these are inefficient in terms of their permeability through the BBB and structural modification of bioactive drugs. In the present study, we therefore examined the usefulness of a noncovalent method using the cell-penetrating peptides (CPPs) such as octaarginine (R8) as a suitable brain delivery strategy for biopharmaceuticals. A safety examination using microvascular endothelial model bEnd.3 cells clarified that R8 was the safest among the CPPs tested in this study. The cellular uptake study demonstrated that coincubation with R8 enhanced the uptake of model peptide drug insulin by bEnd.3 cells in a concentration-dependent and a temperature-independent manner. Furthermore, an in vivo study with rats showed that the accumulation of insulin in the deeper region of the brain, i.e., hippocampus, significantly increased after the intravenous coadministration of insulin with D-R8 without altering the insulin disposition in plasma. Thus, the present study provided the first evidence suggesting that the noncovalent method with CPPs is one of the strategic options for brain delivery of biopharmaceuticals via intravenous injection.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Oligopéptidos/administración & dosificación , Animales , Transporte Biológico , Proteínas Portadoras/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacocinética , Productos del Gen tat/química , Hipoglucemiantes/sangre , Hipoglucemiantes/farmacocinética , Insulina/sangre , Insulina/farmacocinética , Masculino , Ratones , Oligopéptidos/farmacocinética , Ratas Sprague-Dawley , Distribución TisularRESUMEN
This present study aimed to determine the optimal oral insulin delivery conditions that would maximize the utility of cell-penetrating peptides (CPPs) by using a noncovalent strategy. We first compared the effectiveness of two potential CPPs, penetratin and its analog PenetraMax, as absorption enhancers for insulin. The combined effect was evaluated under in vivo oral administration conditions. Both D-forms of CPPs were highly effective for increasing the oral absorption of insulin, and D-PenetraMax showed a more rapid onset of absorption enhancement effects compared with those of D-penetratin. However, synergistic absorption enhancement effects after combination treatment were not observed. Next, we tried a theoretical approach to establish optimized oral insulin delivery conditions. A surface plasmon resonance (SPR)-based analysis demonstrated that binding between insulin and penetratin (2 mM) might be saturated at 100-500 µM penetratin, while the bound concentration of penetratin could increase in accordance with an increased concentration of mixed insulin. To test this hypothesis, we investigated the effectiveness of different insulin doses in the gastric pH-neutralized mice. The results showed that the dissociation of noncovalent complexes of insulin and CPPs at the low gastric pH was prevented in these mice. Our findings clearly suggested that a noncovalent strategy with CPPs represents an effective approach for the L-form of CPP to increase the concentration of CPP-bound insulin to attain greater absorption of insulin, although this approach may not be appropriate for the D-form of CPP. Our findings will contribute to the development of oral dosage forms of insulin for noncovalent strategies involving CPP.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Sistemas de Liberación de Medicamentos , Hipoglucemiantes/administración & dosificación , Insulina Regular Humana/administración & dosificación , Absorción Intestinal/efectos de los fármacos , Administración Oral , Animales , Animales no Consanguíneos , Disponibilidad Biológica , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/química , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Famotidina/farmacología , Ácido Gástrico/química , Ácido Gástrico/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Antagonistas de los Receptores H2 de la Histamina/farmacología , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Insulina Regular Humana/genética , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/farmacología , Ligandos , Masculino , Ratones , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Estereoisomerismo , Resonancia por Plasmón de SuperficieRESUMEN
Ischemic heart disease is a leading cause of death in human population and protection of myocardial infarction (MI) associated with ischemia-reperfusion (I/R) remains a challenge. MG53 is an essential component of the cell membrane repair machinery that protects injury to the myocardium. We investigated the therapeutic value of using the recombinant human MG53 (rhMG53) protein for treatment of MI. Using Langendorff perfusion of isolated mouse heart, we found that I/R caused injury to cardiomyocytes and release of endogenous MG53 into the extracellular solution. rhMG53 protein was applied to the perfusion solution concentrated at injury sites on cardiomyocytes to facilitate cardioprotection. With rodent models of I/R-induced MI, we established the in vivo dosing range for rhMG53 in cardioprotection. Using a porcine model of angioplasty-induced MI, the cardioprotective effect of rhMG53 was evaluated. Intravenous administration of rhMG53, either prior to or post-ischemia, reduced infarct size and troponin I release in the porcine model when examined at 24h post-reperfusion. Echocardiogram and histological analyses revealed that the protective effects of rhMG53 observed following acute MI led to long-term improvement in cardiac structure and function in the porcine model when examined at 4weeks post-operation. Our study supports the concept that rhMG53 could have potential therapeutic value for treatment of MI in human patients with ischemic heart diseases.
Asunto(s)
Cardiotónicos/farmacología , Proteínas Portadoras/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Proteínas Recombinantes/farmacología , Animales , Cardiotónicos/administración & dosificación , Cardiotónicos/farmacocinética , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/farmacocinética , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Técnicas In Vitro , Masculino , Ratones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/mortalidad , Miocardio/metabolismo , Miocardio/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Transducción de Señal/efectos de los fármacos , Porcinos , Proteínas de Motivos TripartitosRESUMEN
PURPOSE: To investigate the suitability of three antimicrobial peptides (AMPs) as cell-penetrating antimicrobial peptides. METHODS: Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cell-penetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in HeLa WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition were analyzed. RESULTS: AMP uptake relative to penetratin was ~13% (PK-12-KKP), ~66% (SA-3) and ~50% (TPk). All four peptides displayed a punctate uptake pattern in HeLa WT cells with co-localization to lysosomes and no indication that clathrin-mediated endocytosis was the predominant uptake mechanism. TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. CONCLUSION: PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Portadoras/farmacología , Péptidos de Penetración Celular/farmacología , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacocinética , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacocinética , Infecciones Bacterianas/tratamiento farmacológico , Proteínas Portadoras/química , Proteínas Portadoras/farmacocinética , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Endocitosis , Células HeLa , Humanos , Datos de Secuencia MolecularRESUMEN
Intraocular drug delivery is extraordinarily hampered by the impermeability of defensive barriers of the eye. In this study, the ocular permeability of fluorophore-labeled cell-penetrating peptides (CPPs), including penetratin, TAT, low molecular weight protamine, and poly(arginine)8, was investigated based on multilevel evaluations. The human conjunctival epithelial cell (NHC) was exposed to various CPPs to determine the cytotoxicity and cellular uptake. Ex vivo studies with rabbit cornea were performed using side-by-side diffusion chambers to evaluate the apparent permeability coefficients and acute tissue tolerance of the CPP candidates. Among all examined CPPs, penetratin shows an outstanding cellular uptake, by increasing more than 16 and 25 times at low and high concentrations, compared to the control peptide poly(serine)8 respectively. Additionally, the permeability of penetratin across excised cornea is 87.5 times higher in comparison with poly(serine)8. More importantly, after instilled in the conjunctival sac of rat eyes, fluorophore-labeled penetratin displayed a rapid and wide distribution in both anterior and posterior segment of the eye, and could be observed in the corneal epithelium and retina lasting for at least 6 h. Interestingly, penetratin showed the lowest ocular cell and tissue toxicities among all examined CPPs. The high ocular permeability of penetratin could be attributed to its amphipathicity and spatial conformation determined by circular dichroism. Taken together, these data demonstrate that penetratin is potentially useful as an absorption enhancer for intraocular drug delivery.
Asunto(s)
Proteínas Portadoras/administración & dosificación , Sistemas de Liberación de Medicamentos , Absorción Ocular/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Péptidos de Penetración Celular , Células Cultivadas , Dicroismo Circular , Córnea/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Permeabilidad , Conejos , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Simulación por Computador , Eritrocitos/efectos de los fármacos , Estradiol/análogos & derivados , Estrenos/farmacología , Sulfonamidas/farmacología , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Anhidrasa Carbónica II/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacocinética , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Descubrimiento de Drogas , Eritrocitos/ultraestructura , Estradiol/farmacocinética , Estradiol/farmacología , Estradiol/toxicidad , Estrenos/farmacocinética , Femenino , Hemólisis/efectos de los fármacos , Humanos , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Investigación Cualitativa , Sulfonamidas/farmacocinética , Sulfonamidas/toxicidad , TemperaturaRESUMEN
Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man alpha(1-2)Man, Man alpha(1-2)Man alpha(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K(d) = 3.4 x 10(-8) M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high- and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the "cluster effect" of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.
Asunto(s)
Proteínas Bacterianas/farmacología , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Lectinas/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacocinética , Sitios de Unión , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Cristalografía por Rayos X , Proteína gp120 de Envoltorio del VIH/química , Inhibidores de Fusión de VIH/química , Inhibidores de Fusión de VIH/farmacocinética , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Técnicas In Vitro , Cinética , Lectinas/química , Lectinas/farmacocinética , Manosa/química , Manósidos/química , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
The interest of the scientific community for cell penetrating peptides (CPP) has been growing exponentially for these last years, and the list of novel CPP is increasing. These peptides are powerful tools for the delivery of cargoes to their site of action. Indeed, several drugs that cannot translocate through the cell plasma membrane have been successfully delivered into cells when grafted to a CPP. Various cargoes have been linked to CPP, such as oligonucleotides, pharmacologically active drugs, contrast agents for imaging, or nanoparticles as platforms for multigrafting purposes This review illustrates the fabulous potential of CPP and the diversity of their use, but their most interesting application appears their future clinical use for the treatment of various pathological conditions.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Portadores de Fármacos/administración & dosificación , Sistemas de Liberación de Medicamentos , Secuencia de Aminoácidos , Animales , Antineoplásicos/administración & dosificación , Transporte Biológico , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/farmacocinética , Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular/farmacocinética , Portadores de Fármacos/farmacocinética , Endocitosis , Colorantes Fluorescentes/administración & dosificación , Productos del Gen tat/administración & dosificación , Productos del Gen tat/farmacocinética , Humanos , Modelos Biológicos , Imagen Molecular/métodos , Datos de Secuencia Molecular , Nanopartículas/administración & dosificación , Péptidos/administración & dosificación , Péptidos/farmacocinética , Venenos de Escorpión/administración & dosificación , Venenos de Escorpión/farmacocinéticaRESUMEN
We have evaluated the utility of epitope binning on biolayer interferometry (BLI) as a strategy to funnel the selection of candidate pairs suitable for pharmacokinetic assay development. Totally, 8 anti-Idiotypic monoclonal antibodies in 64 possible combinations were tested by BLI, ELISA and Gyrolab®. Two epitope binning approaches were utilized, in-tandem and classic sandwich. Both formats identified four mutually exclusive bins providing 31 and 25 possible antibody pair combinations, respectively. In contrast, the ELISA and Gyrolab yielded 18 and 9 positive pairs, respectively, with only a partial correlation to the BLI results. Several positive pairs by ELISA and Gyrolab, screened negative by BLI. Just over half of the pairs predicted by BLI were positive on ELISA and less than a quarter were positive on Gyrolab. This evaluation showed, in our case, that BLI was limited in its ability to predict candidate pairs that would be successful in pharmacokinetic method development.
Asunto(s)
Proteínas Portadoras/farmacocinética , Indicadores y Reactivos/química , Ligandos , HumanosRESUMEN
UNLABELLED: Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation. METHODS: In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, (68)Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model. RESULTS: The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that (68)Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of (68)Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo. CONCLUSION: This proof-of-concept research clearly demonstrated that synthetic 2-helix (68)Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.
Asunto(s)
Proteínas Portadoras/farmacocinética , Radioisótopos de Galio/farmacocinética , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/metabolismo , Tomografía de Emisión de Positrones/métodos , Receptor ErbB-2/metabolismo , Animales , Proteínas Portadoras/química , Línea Celular Tumoral , Femenino , Radioisótopos de Galio/química , Perfilación de la Expresión Génica/métodos , Humanos , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Especificidad de Órganos , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
*Romiplostim is an Fc-peptide fusion protein (or 'peptibody') that stimulates megakaryopoiesis and thrombopoiesis by binding to, and activating, the thrombopoietin receptor. *Because it has no sequence homology to thrombopoietin, romiplostim theoretically avoids the risk of eliciting cross-reacting, neutralizing antibodies to thrombopoietin. *In well designed, 24-week, phase III trials, subcutaneous romiplostim was significantly more effective than placebo in achieving the primary endpoint of a protocol-defined durable platelet response in nonsplenectomized (61% vs 5%) or splenectomized (38% vs 0%) adults with chronic immune (idiopathic) thrombocytopenic purpura (ITP). *Romiplostim was also significantly more effective than placebo with regard to a number of secondary endpoints, including the proportions of patients with an overall (durable plus transient) platelet response or who required ITP rescue medications. The majority of romiplostim-treated patients receiving concurrent ITP drugs were able to reduce or discontinue these therapies. *Platelet response was maintained by most patients during longer-term treatment with romiplostim for up to 3 or 4 years in an open-label extension study. *Romiplostim was generally well tolerated. Almost all adverse events in the phase III studies were of mild-to-moderate intensity; most were unrelated to treatment. Longer-term treatment with romiplostim had an adverse event profile consistent with that observed in the phase III studies.
Asunto(s)
Proteínas Portadoras , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Receptores Fc , Proteínas Recombinantes de Fusión , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Proteínas Portadoras/uso terapéutico , Enfermedad Crónica , Ensayos Clínicos Fase III como Asunto , Humanos , Infusiones Subcutáneas , Receptores Fc/uso terapéutico , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , TrombopoyetinaRESUMEN
The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Oligonucleótidos Antisentido/farmacocinética , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/síntesis química , Proteínas Portadoras/genética , Línea Celular Transformada , Péptidos de Penetración Celular , Dicroismo Circular , Regulación hacia Abajo , Portadores de Fármacos/síntesis química , Técnicas de Transferencia de Gen , Ratones , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Conformación Proteica , Transporte de Proteínas , Proteína EWS de Unión a ARNRESUMEN
OBJECTIVE: To review the pharmacology, pharmacokinetics, efficacy, and safety of romiplostim, the first drug approved for use in patients with immune thrombocytopenic purpura (ITP). DATA SOURCES: Articles were identified through searches of MEDLINE (1966-January 2009) and International Pharmaceutical Abstracts (1970-January 2009) using the key words romiplostim and AMG 531. Searches were limited to articles published in English. The manufacturer was contacted for additional data. STUDY SELECTION AND DATA EXTRACTION: Clinical trials and pharmacokinetic data were selected for review. DATA SYNTHESIS: Romiplostim is a second-generation thrombopoietic receptor agonist that exerts its therapeutic effect by stimulating megakaryopoiesis. Subcutaneous therapy results in a dose-dependent increase in platelets; however, interindividual variability exists. Time to peak concentration is approximately 14 hours, and the elimination half-life is approximately 3.5 days (range 1-34). Romiplostim undergoes endothelial recirculation and is eliminated by the reticuloendothelial system. The results of 2 Phase 3, randomized, double-blind, placebo-controlled trials have demonstrated the efficacy of romiplostim for increasing platelet counts in patients with ITP refractory to other therapies, including splenectomy. Effects on platelets were transient and decreased within 2 weeks of discontinuing the drug. Interim results of an open-label extension study revealed that romiplostim has sustained efficacy and tolerability for up to 156 weeks at a dosage range of 1-17 microg/kg/wk (mean 5.9 +/- 3.9). The most common adverse effects include headache, fatigue, epistaxis, and contusion. Romiplostim is also under investigation for treatment of thrombocytopenia associated with myelodysplastic syndrome. The drug must be ordered directly from the manufacturer through a limited access program, and weekly subcutaneous injections are given in the clinic setting. CONCLUSIONS: Romiplostim is effective for the management of ITP in adults refractory to other therapies, including splenectomy.
Asunto(s)
Proteínas Portadoras , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Receptores Fc , Proteínas Portadoras/efectos adversos , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Proteínas Portadoras/uso terapéutico , Ensayos Clínicos como Asunto , Esquema de Medicación , Costos de los Medicamentos , Humanos , Receptores Fc/uso terapéutico , Proteínas Recombinantes de Fusión , TrombopoyetinaRESUMEN
Acute intoxication with organophosphates (OPs) can trigger status epilepticus followed by persistent cognitive impairment and/or electroencephalographic abnormalities. Neuroinflammation is widely posited to influence these persistent neurological consequences. However, testing this hypothesis has been challenging, in part because traditional biometrics preclude longitudinal measures of neuroinflammation within the same animal. Therefore, we evaluated the performance of noninvasive positron emission tomography (PET), using the translocator protein (TSPO) radioligand [18F]PBR111 against classic histopathologic measures of neuroinflammation in a preclinical model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im) and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. TSPO PET performed prior to DFP exposure and at 3, 7, 14, 21, and 28 days postexposure revealed distinct lesions, as defined by increased standardized uptake values (SUV). Increased SUV showed high spatial correspondence to immunohistochemical evidence of neuroinflammation, which was corroborated by cytokine gene and protein expression. Regional SUV metrics varied spatiotemporally with days postexposure and correlated with the degree of neuroinflammation detected immunohistochemically. Furthermore, SUV metrics were highly correlated with seizure severity, suggesting that early termination of OP-induced seizures may be critical for attenuating subsequent neuroinflammatory responses. Normalization of SUV values to a cerebellar reference region improved correlations to all outcome measures and seizure severity. Collectively, these results establish TSPO PET using [18F]PBR111 as a robust, noninvasive tool for longitudinal monitoring of neuroinflammation following acute OP intoxication.
Asunto(s)
Proteínas Portadoras/farmacocinética , Inflamación/diagnóstico por imagen , Isoflurofato/toxicidad , Síndromes de Neurotoxicidad/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Quimiocinas/análisis , Citocinas/genética , Radioisótopos de Flúor , Inflamación/inducido químicamente , Inflamación/inmunología , Masculino , Síndromes de Neurotoxicidad/inmunología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-ARESUMEN
Possibility to predict short peptide sequences capable to penetrate the plasma membrane opens new opportunities for developing peptide based intracellular delivery vectors, called cell-penetrating peptides (CPPs). Predictions of CPPs, however are often based on trial and error and may not always lead to new potent sequences. In this review we discuss different problems associated with CPP prediction. Additionally, the used methods of CPP prediction are compared. Also, a few suggestions are made for designing new CPP sequences and improvement of predictions.
Asunto(s)
Proteínas Portadoras/química , Péptidos/química , Algoritmos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/farmacocinética , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Sistemas de Liberación de Medicamentos/métodos , Humanos , Datos de Secuencia Molecular , Péptidos/administración & dosificación , Péptidos/farmacocinéticaRESUMEN
Most of this volume is dedicated to a very important and pragmatic issue which is to design ways of internalizing active pharmacological compounds into cells. In fact, many vectors have now been developed and the improvement in the technology can be seen on two main fronts. A first one is the identification of extremely efficient cargoes, for example siRNAs, which can enter the cells once attached to the vectors. A second one is the development of chemical vectors designed after the properties of the peptides and of tags allowing in vivo addressing to specific organs, for example the brain, cell types or sub-cellular compartments. This chapter is of a different nature, as it is devoted to the physiological significance of protein transduction and to the comparative analysis of the Penetratin PTD with its parental proteins, the homeoproteins. Although very academic, these two issues are of practical interest for the rational design of new vectors and the identification of unforeseen pathological mechanisms and pharmacological targets.